Anti-ccl17 antibodies

ABSTRACT

The present invention relates to antibodies specifically binding CCL17, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 15/835,529, filed Dec. 8,2017, which is a divisional of application Ser. No. 14/534,525, filedNov. 6, 2014, which claims the benefit of U.S. Provisional ApplicationNo. 61/900,596, filed Nov. 6, 2013. The contents of these applicationsare each incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies specifically binding CCL17,polynucleotides encoding the antibodies or fragments, and methods ofmaking and using the foregoing.

BACKGROUND OF THE INVENTION

The homeostatic chemokine, CCL17 (TARC, chemokine (C-C motif) ligand 17)is a potent lymphocyte chemoattractant. CCL17 is a ligand for CCR4, aGPCR believed to be important in the function of T cells and chemotaxisand the migration of immune cells to sites of inflammation. CCR4 ispredominantly expressed on Th2 lymphocytes, natural killer cells andiNKT cells. CCL17 has been associated with human diseases affectingvarious organs such as ulcerative colitis (UC), atopic dermatitis (AD),idiopathic pulmonary fibrosis (IPF) and asthma (Belperio et al., JImmunol, 173: 4692-469, 2004; Christophi et al., Inflamm Bowel Dis. doi:10.1002/ibd.2295; Inoue et al., Eur Respir J, 24: 49-56, 2004; Kakinumaet al., J Allergy Clin Immunol, 107: 535-541, 2001; Saeki and Tamaki, JDermatol Sci, 43: 75-84, 2006; Tamaki et al., J Dermatol, 33: 300-302,2006). In mice, CCL17 has been linked to various inflammatory conditionsand infections such as chronic pulmonary inflammation present in modelsof fibrosis and asthma, colitis and schistosomiasis, presumably byinducing Th2 responses through recruitment of CCR4+ immune cells.Neutralization of CCL17 ameliorates the impacts of disease in both theA. fumigatus and ovalbumin (OVA) models of asthma, and liver damage inthe P. acnes mouse model of induced hepatic injury by blocking influx ofT cells (Carpenter and Hogamoam Infect Immun, 73:7198-7207, 2005;Heiseke et al., Gastroenterology, 142:335-345; Hogamoam et al,, MedMycol, 43 Suppl 1, S197-202, 2005; Ismailoglu et al., Therapeutictargeting of CCL17 via the systemic administration of a monoclonalantibody ameliorates experimental fungal asthma. Paper presented at theAm J Respir Crit Care Med, 2011; Jakubzick et al., Am J Pathol,165:1211-122, 2004; Kawasaki et al., J Immunol, 166:2055-2062, 2001;Yoneyama et al., J Clin Invest, 102:1933-1941, 1998).

CCL22 (MDC) is a second ligand for CCR4. CCR4 interaction with eachchemokine produces distinct outcomes (Allen et al., Annu Rev Immunol25:787-820, 2007; Imai et al., J Biol Chern 273:1764-1768, 1998),possibly contributed by the differences in binding affinities of the twoligands for CCR4. CCL22 binds CCR4 with higher affinity and inducesreceptor internalization more readily than CCL17 (Baatar et al., JImmunol 179:1996-2004, 2007; Imai et al., J Biol Chem 273:1764-1768,1998; Mariani et al., Eur J Immunol 34:231-240, 2004), and promotescellular adhesion more readily than CCL17 (D'Ambrosio et al., J Immunol169:2303-2312, 2002) CCL22 shows more restricted expression withproduction limited to immune cells, whereas CCL17 is expressed andsecreted by many different cell types including non-immune cells(Alferink et al., J Exp Med 197:585-599, 2003; Berin et al., Am J RespirCell Mol Biol 24:382-389, 2001; Godiska et al., J Exp Med 185:1595-1604,1997; Imai et al., J Biol Chem 271:21514-21521, 1996; Saeki and Tamaki,J Bermatol Sci 43:75-84, 2006). In the murine ceacal ligation andpuncture (CLP) model of experimental sepsis, CCL22 promoted innateimmunity whereas CCL17 seemed to interfere and in some circumstancescontribute to organ damage (Matsukawa et al., Rev Immunogenet 2:339-358,2000). In the mouse model of pulmonary invasive aspergillosis, CCL22played a protective role in the innate anti-fungal response whereasCCL17 played the role of suppressor (Carpenter and Hogaboam, InfectImmun 73:7198-7207, 2005). These two chemokines can play contrastingroles in establishing localized inflammation due to differential effectson Treg homeostasis in that Treg recruitment is favored by CCL22 but notCCL17 (Heiseke et

al., Gastroenterology 142:335-345,2011; Montane et al., J Clin Invest,121:3024-30, 2011; Weber et al., J Clin Invest 121:2898-2910, 2011).

In animal model of contact hypersensitivity, CCL17 is a major factor ininitiating the inflammatory response driving contact hypersensitivity(CBS) to challenge with either FITC or DNFB, and CCL17 knockout in thesemice enhanced survival of cardiac allografts compared to heterozygousmice having one functional CCL17 allele (Alferink et al., J Exp Med197:585-599, 2003).

CCR4 antagonists may be non-selective and inhibit both CCL17 and CCL22functions. Therefore, there is a need for anti-CCL17 antibodies for thepotential treatment of a variety of CCL17-mediated diseases includingasthma.

SUMMARY OF THE INVENTION

One embodiment of the invention is an isolated antibody specificallybinding human CCL17 comprising a heavy chain variable region (VH) and alight chain variable region (VL), wherein the antibody competes forbinding to human CCL17 with an antibody comprising the VH of SEQ IDNO:45 and the VL of SEQ ID NO: 52.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17 having the sequence of SEQ ID NO: 1, wherein theantibody binds human CCL17 at least within CCL17 amino acid residues21-23, 44-45 and 60-68.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17, wherein the antibody binds human CCL17 with anaffinity constant (K_(D)) of about 1×10⁻¹⁰ M or less, when the K_(D) ismeasured using solution equilibrium affinity in tris-based saline buffercontaining 0.05% Tween-20 after co-incubation of the antibody and humanCCL17 for 48 hours at 4° C.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17 comprising certain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3 sequences.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17 comprising certain VH and VL sequences.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17, wherein the antibody comprises the VH comprisingthe amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the VH of SEQ ID NO: 46 and the VL comprisingthe amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the VL of SEQ ID NO: 62.

Another embodiment of the invention is a pharmaceutical compositioncomprising the antibody of the invention and a pharmaceutically acceptedcarrier.

Another embodiment of the invention is an isolated polynucleotideencoding the VH or the VL of the invention.

Another embodiment of the invention is a vector comprising thepolynucleotide of the invention.

Another embodiment of the invention is a host cell comprising the vectorof the invention.

Another embodiment of the invention is a method of producing an antibodyof the invention, comprising culturing the host cell of the invention inconditions that the antibody is produced.

Another embodiment of the invention is a method of treating aCCL17-mediated disease, comprising administering to a subject in needthereof the antibody of the invention for a time sufficient to treat theCCL17-mediated disease.

Another embodiment of the invention is a method of treating asthma orairway hyper-reactivity, comprising administering to a subject theantibody of the invention for a time sufficient to treat asthma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows inhibition of chemotaxis with anti-CCL17 antibody 8302induced by 1 nM human CCL17 in CCRF-CEM cells. 8302 is C178302.

FIG. 1B shows inhibition of chemotaxis with anti-CCL17 antibody 8311induced by 1 nM human CCL17 in CCRF-CEM cells. 8311 is C178311.

FIG. 1C shows effect of IgG2 isotype control on chemotaxis induced by 1nM human CCL17 in CCRF-CEM cells.

FIG. 1D shows effect of IgG4 isotype control on chemotaxis induced by 1nM human CCL17 in CCRF-CEM cells.

FIG. 2A shows inhibition of chemotaxis with anti-CCL17 antibody 8302induced by 1 nM cyno CCL17 in HSC-F cells. 8302 is C178302.

FIG. 2B shows inhibition of chemotaxis with anti-CCL17 antibody 8311induced by 1 nM cyno CCL17 in HSC-F cells. 8311 is C178311.

FIG. 2C shows effect of IgG2 isotype control on chemotaxis induced by 1nM cyno CCL17 in HSC-F cells.

FIG. 2D shows effect of IgG4 isotype control on chemotaxis induced by 1nM cyno CCL17 in HSC-F cells.

FIG. 3 shows the VH sequences of the anti-CCL17 antibodies binding tohuman CCL17 with a K_(D) of 100 nM or lower. C17B234VH: SEQ ID NO: 45;C17B235VH: SEQ ID NO: 45; C17B236VH: SEQ ID NO: 45; C17B239VH: SEQ IDNO: 45; C17B240VH: SEQ ID NO: 45; C17B241VH: SEQ ID NO: 45; C17B243VH:SEQ ID NO: 45; C17B244VH: SEQ ID NO: 45; C17B293VH: SEQ ID NO: 46;C17B294VH: SEQ ID NO: 47.

FIG. 4 shows the consensus VH and HCDR sequences of the anti-CCL17antibodies shown in FIG. 3 that bind to human CCL17 with a KD of 100 nMor lower.

FIG. 5 shows the VL sequences of the anti-CCL17 antibodies binding tohuman CCL17 with a K_(D) of 100 nM or lower. C17B234VL: SEQ ID NO: 50;C17B235VL: SEQ ID NO: 51; C17B236VL: SEQ ID NO: 52; C17B239VL: SEQ IDNO: 55; C17B240VL: SEQ ID NO: 56; C17B241VL: SEQ ID NO: 57; C17B243VL:SEQ ID NO: 59; C17B244VL: SEQ ID NO: 60; C17B293VL: SEQ ID NO: 62;C17B294VL: SEQ ID NO: 62.

FIG. 6A shows the consensus VL sequence of the anti-CCL17 antibodiesshown in FIG. 5 that bind to human CCL17 with a K_(D) of 100 nM orlower.

FIG. 6B shows the consensus LCDR sequences of the anti-CCL17 antibodiesshown in FIG. 5 that bind to human CCL17 with a K_(D) of 100 nM orlower.

FIG. 7 shows the epitope and paratope residues of antibody C17B236. VHand VL paratope residues are boxed, and CCL17 epitope residues arecircled. Residue numbering is according to SEQ ID NO: 45 (VH), SEQ IDNO: 52 (VL), SEQ ID NO: 1 (CCL17).

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentinvention, exemplary materials and methods are described herein. Indescribing and claiming the present invention, the following terminologywill be used.

“Specific binding” or “specifically binds” or “binds” as used hereinrefers to antibody binding to a predetermined antigen with greateraffinity than for other antigens. Typically, the antibody binds to apredetermined antigen with a dissociation constant (K_(D)) of about1×10⁻⁷ M or less, for example about 1×10⁻⁸ M or less, about 1×10⁻⁹ M orless, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ M or less, about 1×10⁻¹² Mor less, about 1×10⁻¹³ M or less or about 1×10⁻¹⁴ M or less, typicallywith a K_(D) that is at least ten fold less than its K_(D) for bindingto a non-specific antigen or epitope (e.g., BSA, casein). Thedissociation constant can be measured using standard procedures.Antibodies that specifically bind to a predetermined antigen may,however, have cross-reactivity to other related antigens, for example tothe same predetermined antigen from other species (homologs), such ashuman or monkey, for example Macaca fascicularis (cynomolgus, cyno) orPan troglodytes (chimpanzee, chimp).

“Monoclonal antibody that specifically binds human CCL17” refers toantibodies that specifically bind human mature CCL17 having the sequenceshown in SEQ ID NO: 1.

“Neutralizing” or “neutralizes” or “neutralizing antibody” or “antibodyantagonist” as used herein refers to an antibody or antibody fragmentthat partially or completely inhibits, by any mechanism, CCL17biological activity. Neutralizing antibodies can be identified usingassays for CCL17 biological activity as described below. CCL17neutralizing antibody may inhibit measured CCL17 biological activity by20%, 30%, 40%, 50%, 60%, 70%, 75%, 90%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100%.

“Human CCL17” or “huCCL17” as used interchangeably herein refers to thehuman CCL17 protein having the amino acid sequence shown in SEQ IDNO: 1. The sequence of the full length CCL17 including the signalsequence is available at GenBank; Accession Number NP_002978.

“Cyno CCL17” or “cCCL17” as used interchangeably herein refers to theMacaca fascicularis (cyno) CCL17 protein having the amino acid sequenceshown in SEQ ID NO: 2.

“Antibodies” as used herein is meant in a broad sense and includesimmunoglobulin molecules including monoclonal antibodies includingmurine, human, humanized and chimeric antibodies, antibody fragments,bispeciric or multispecific antibodies formed from at least two intactantibodies or antibody fragments, dimeric, tetrameric or multimericantibodies, single chain antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD,IgE, IgG and IgM, depending on the heavy chain constant domain aminoacid sequence. IgA and IgG are further sub-classified to IgA₁, IgA₂,IgG₁, IgG₂, IgG₃ and IgG₄ isotypes. Antibody light chains of anyvertebrate species can be assigned to one of two clearly distinct types,namely kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

The term “antibody fragments” refers to a portion of an immunoglobulinmolecule that retains the heavy chain and/or the light chain antigenbinding site, such as s heavy chain complementarity determining regions(HCDR) 1, 2 and 3, a light chain complementarity determining regions(LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chainvariable region (VL). Antibody fragments include a Fab fragment, amonovalent fragment consisting of the VL or the VH; a F(ab)₂fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; a Fd fragment consisting of the VH and CHIdomains; a Fv fragment consisting of the VL and VH domains of a singlearm of an antibody; a dAb fragment (Ward et al., Nature 341:544-546,1939), which consists of a VH domain. VH and VL domains can beengineered and linked together via a synthetic linker to form varioustypes of single chain antibody designs where the VH/VL domains pairintramolecularly, or intermoiecuiarly in those cases when the VH and VLdomains are expressed by separate single chain antibody constructs, toform a monovalent antigen binding site, such as single chain Fv (scFv)or diabody; described for example in Int. Pat. Publ. No. WO1998/44001,Int. Pat. Publ. No. WO1938/01649; Int. Pat. Publ. No. WO1994/13804; Int.Pat. Publ. No. WO1992/01047. These antibody fragments are obtained usingwell known techniques and the fragments are characterized in the samemanner as are intact antibodies.

The phrase “isolated antibody” refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody specifically binding humanCCL17 is substantially free of antibodies that specifically bindantigens other than human CCL17). An isolated antibody specificallybinding human CCL17 may, however, have cross-reactivity to otherantigens, such as orthologs of human CCL17, such as Macaca fascicularis(cynomolgus) CCL17. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals.

An antibody variable region consists of a “framework” region interruptedby three “antigen binding sites”. The antigen binding sites are definedusing various terms: (i) Complementarity Determining Regions (CDRs),three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2,LCDR3) are based on sequence variability (Wu and Kabat, J Exp Med132:211-50, 1970; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991). (ii) “Hypervariable regions”, “HVR”, or “HV”,three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer tothe regions of an antibody variable domains which are hypervariable instructure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol196:901-17, 1987). Other terms include “IMGT-CDRs” (Lefranc et al., DevComparat Immunol 27:55-77, 2003) and “Specificity Determining ResidueUsage” (SDRU) (Almagro Mol Recognit, 17:132-43, 2004). The InternationalImMunoGeneTics (IMGT) database provides a standardized numbering anddefinition of antigen-binding sites. The correspondence between CDRs,HVs and IMGT delineations is described in Lefranc et al., Dev ComparatImmunol 27:55-77, 2003.

“Chothia residues” as used herein are the antibody VL and VH residuesnumbered according to Al-Lazikani (Al-Lazikani et al., J Mol Biol273:927-48, 1997).

“Framework” or “framework sequences” are the remaining sequences of avariable region other than those sequences defined to be antigen bindingsite. Because the antigen binding site can be defined by various termsas described above, the exact amino acid sequence of a framework dependson how the antigen-binding site was defined.

“Humanized antibody” refers to an antibody in which the antigen bindingsite is derived from non-human species and the variable regionframeworks are derived from human immunoglobulin sequences. Humanizedantibodies may include substitutions in the framework regions so thatthe framework may not be an exact copy of expressed human immunoglobulinor germline gene sequences.

“Human antibody” refers to an antibody having heavy and light chainvariable regions in which both the framework and the antigen bindingsite regions are derived from sequences of human origin. If the antibodycontains a constant region, the constant region also is derived fromsequences of human origin.

Human antibody comprises heavy or light chain variable regions that are“derived from” sequences of human origin if the variable regions of theantibody are obtained from a system that uses human germlineimmunoglobulin or rearranged immunoglobulin genes. Such systems includehuman immunoglobulin gene libraries, for example libraries displayed onphage, and transgenic non-human animals such as mice carrying humanimmunoglobulin loci as described herein. “Human antibody” may containamino acid differences when compared to the human germline or rearrangedimmunoglobulin sequences due to for example naturally occurring somaticmutations or intentional introduction of substitutions. Typically,“human antibody” is at least about 80%, 35%, 90%, 95%, 96%, 97%, 98%,99% or 100% identical in amino acid sequence to an amino acid sequenceencoded by a human germline or rearranged immunoglobulin gene. In somecases, “human antibody” may contain consensus framework sequencesderived from human framework sequence analyses, for example as describedin Knappik et al., J Mol Biol 296:57-86, 2000), or synthetic HCDR3incorporated into human immunoglobulin gene libraries displayed onphage, for example as described in Shi et al., J Mol Biol 397:385-96,2010 and Int. Pat. Publ. No. WO2009/08546).

Isolated humanized antibodies may be synthetic. Human antibodies, whilederived from human immunoglobulin sequences, can be generated usingsystems such as phage display incorporating synthetic CDRs and/orsynthetic frameworks, or can be subjected to in vitro mutagenesis toimprove antibody properties, resulting in antibodies that do notnaturally exist within the human antibody germline repertoire in vivo.

Human antibodies may include substitutions in the framework or in theantigen binding site so that they may not be exact, copies of expressedhuman immunoglobulin or germline gene sequences. However, antibodies inwhich antigen binding sites are derived from a non-human species are notincluded in the definition of “human antibody”.

The term “recombinant antibody” as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal for human immunoglobulin genes or ahybridoma prepared therefrom (described further below), antibodiesisolated from a host cell transformed to express the antibody,antibodies isolated from a recombinant, combinatorial antibody library,and antibodies prepared, expressed, created or isolated by any othermeans that, involve splicing of human immunoglobulin gene sequences toother DNA sequences.

The term “monoclonal antibody” as used herein refers to a preparationof: antibody molecules of single molecular composition.

The term “substantially identical” as used herein means that the twoantibody variable region amino acid sequences being compared areidentical or have “insubstantial differences”. Insubstantial differencesare substitutions of 1, 2, 3, 4, 5, 6, 7, 3, 9, 10, 11, 12, 13, 14, or15 amino acids in an antibody variable region sequence that do notadversely affect antibody properties. Amino acid sequences substantiallyidentical to the variable region sequences disclosed herein are withinthe scope of the invention. In some embodiments, the sequence identitycan be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.Percent identity can be determined for example by pairwise alignmentusing the default settings of the AlignX module of Vector NTI v.9.0.0(Invitrogen, Carlsbad, Calif.). The protein sequences of the presentinvention can be used as a query sequence to perform a search againstpublic or patent databases to, for example, identify related sequences.Exemplary programs used to perform such searches are the XBLAST orBLASTP programs, or the GenomeQuest™ (GenomeQuest, Westhorough, Mass.)suite using the default settings.

The term “epitope” as used herein means a portion of an antigen to whichan antibody specifically binds. Epitopes usually consist of chemicallyactive (such as polar, non-polar or hydrophobic) surface groupings ofmoieties such as amino acids or polysaccharide side chains and can havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An epitope can be composed ofcontiguous and/or discontiguous amino acids that form a conformationalspatial unit. For a discontiguous epitope, amino acids from differingportions of the linear sequence of the antigen come in close proximityin 3-dirnensional space through the folding of the protein molecule.

“Bispecific” as used herein refers to an antibody or molecule that bindstwo distinct antigens or two distinct epitopes within an antigen.

“Monospecific” as used herein refers to an antibody that binds oneantigen or one epitope.

The term “in combination with” as used herein means that the describedagents can be administered to an animal together in a mixture,concurrently as single agents or sequentially as single agents in anyorder.

The term “vector” means a non-natural polynucleotide capable of beingduplicated within a biological system or that can be moved between suchsystems. Vector polynucleotides typically contain a cDNA encoding aprotein of interest and additional elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

“Complementary DNA” or “cDNA” refers to a well known syntheticpolynucleotide that shares the arrangement of sequence elements found innative mature mRNA species with contiguous exons, with the interveningintrons present in genomic DNA are removed. The codons encoding theinitiator methionine may or may not be present in cDNA. cDNA may besynthesized for example by reverse transcription or synthetic geneassembly.

“Synthetic” or “non-natural” as used herein refers to a polynucleotideor a polypeptide molecule not present in nature.

The term “polypeptide” or “protein” means a molecule that comprises atleast two amino acid residues linked by a peptide bond to form apolypeptide. Small polypeptides of less than 50 amino acids may bereferred to as “peptides”.

Conventional one and three-letter amino acid codes are used herein asshown in Table 1.

TABLE 1 Amino Three-letter One-letter Acid code code Alanine Ala AArginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C GlutamateGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F PralinePro P Serine Ser s Threonine Thr T Tryptophan Trp w Tyrosine Tyr YValine Val V

Compositions of Matter

The present invention provides monoclonal antibodies specificallybinding human CCL17. The antibodies of the invention inhibit CCL17biological activity in the cell, and may optionally cross-react withcyno CCL17. The present invention provides synthetic polynucleotidesencoding the antibodies and fragments thereof, vectors and host cells,and methods of making and using the antibodies of the invention.

One embodiment of the invention is an isolated antibody specificallybinding human CCL17.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17 comprising a heavy chain variable region (VH) and alight chain variable region (VL), wherein the antibody competes forbinding to human CCL17 with an antibody comprising the VH of SEQ IDNO:45 and the VL of SEQ ID NO: 52. The antibody comprising the VH of SEQID NO: 46 and the VL of SEQ ID NO: 62 is expected to compete for bindingto human CCL17 with the antibody comprising the VH of SEQ ID NO: 45 andthe VL of SEQ ID NO: 52.

Competition between specific binding to human CCL17 with antibodies ofthe invention comprising certain VH and VL amino acid sequences can beassayed in vitro using well known methods. For example, binding of MSDSulfa-Tag™ NBS-ester-labeled antibody to human CCL17 in the presence ofan unlabeled antibody can be assessed by ELISA, or Biacore analyses orflow cytometry may be used to demonstrate competition with theantibodies of the current invention. The ability of a test antibody toinhibit the binding of the antibody comprising the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 52 to human CCL17 demonstrates that the testantibody can compete with these antibodies for binding to human CCL17.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17 having the sequence of SEQ ID NO: 1, wherein theantibody binds human CCL17 at least within CCL17 amino acid residues21-23, 44-45 and 60-68. “At least within human CCL17 amino acid residues21-23, 44-45 and 60-68” means that the anti-CCL17 antibody binds atleast one residue residing within the amino acid stretch of residues21-23 of SEQ ID NO: 1, and at least one residue residing within theamino acid stretch of residues 44-45 of SEQ ID NO: 1, and at least oneresidue residing within the amino acid stretch of residues 60-68 of SEQID NO: 1. The antibody may bind more than one residue within theresidues 21-23, 44-45 and 60-68, and additional residues outside ofresidues 21-23, 44-45 and 60-68 of SEQ ID NO: 1.

In some embodiments, the antibody binds human CCL17 at least at residuesR22 and K23 of SEQ ID NO: 1.

In some embodiments, the antibody binds human CCL17 at least at residuesL21, R22, K23, V44, Q45, N60, Y64, S67 and L68 of SEQ ID NO: 1.

An exemplary antibody that binds human CCL17 within CCL17 amino acidresidues 21-23, 44-45 and 60-68 of SEQ ID NO: 1 is C17B236 having VH ofSEQ ID NO: 45 and VL of SEQ ID NO: 52. Based on crystal structureanalyses, the main epitope residues bound by C17B236 are R22 and K23 ofCCL17 of SEQ ID NO: 1, based on the number of contacts between theseresidues and the antibody VH residues.

Other exemplary antibodies that bind human CCL17 within CCL17 amino acidresidues 21-23, 44-45 and 60-68 are variants of C17B236, which arederived from an affinity-maturation campaign of the same parentalantibody. The VH and the VL sequences of the exemplary antibodies areshown in FIG. 3 and FIG. 5. The antibody comprising the VH of SEQ ID NO:46 and the VL of SEQ ID NO: 62 is expected to bind to human CCL17 withinCCL17 amino acid residues 21-23, 44-45 and 60-68.

Affinity maturation of antibodies typically involves amino acidsubstitutions in the CDRs or in the Vernier zone (framework regions thatunderline the CDRs). The matured variants are selected by panning thecombinatorial libraries, which may contain up to 10⁸ mutants. The cap onthe size of the library limits the number of variable positions to 6-7if all 20 amino acids are allowed in each position. The majority of theparatope residues are preserved in each combinatorial library, whichensures that the binding epitope is also preserved. Severalcrystallographic studies of the parent and matured antibodies have shownthat the epitope is always preserved during affinity maturation (e.g.Fransson et al., J. Mol. Biol. 2010, 398:214-231; Gustchina et al., PLoSPathog. 2010, 6:e1001182; La Porte et al., MAbs 2014; 6:1059-1068).

Anti-CCL17 antibodies that bind human CCL17 within CCL17 amino acidresidues 21-23, 44-45 and 60-68 bind human CCL17 with high affinity,typically with the K_(D)less than about 1×10⁻¹⁰ M.

Antibodies that bind human CCL17 within CCLI7 amino acid residues 21-23,44-45 and 60-68 may be made for example by immunizing mice with CCL17chimeric protein that has human CCL17 sequences at residue positions21-23, 44-45 and 60-68, or panning phage display libraries with wildtype human CCL17, and cross-screening the resulting hits with CCL17variants that have substitutions at each or several residue positionswithin the residues 21-23, 44-45 and 60-68 of human CCL17 using methodsdescribed herein.

In some embodiments of the invention, the antibody specifically bindinghuman CCL17 blocks CCL17/CCR4 interaction.

Antibodies can be tested for their ability to block CCL17/CCR4interaction by standard flow cytometry. For example, cells expressingCCR4 are incubated with fluorescently labeled human CCL17 and the testantibody, after which the binding of the fluorescently labeled humanCCL17 onto CCR4-expressing cells is assessed using standard methods.Antibodies that “block CCL17/CCR4 interaction” or “inhibit CCL17/CCR4interaction” may inhibit binding of CCL17 to CCR4-expressing cells by30%, 40%, 50%, 60%, 70%, 75%, 80%, 35%, 90%, 95%, 96%, 97%, 98%, 99% or100% when compared to binding of CCL17 in the absence of the antibody.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17, wherein the antibody binds human CCL17 with anaffinity constant (K_(D)) of about 1×10⁻⁷ M or less, of about 1×10⁻⁸ Mor less, about 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹M or less, about 1×10⁻¹² M or less, about 1×10⁻¹³ M or less, or about1×10⁻¹⁴ M or less, when the K_(D) is measured using solution equilibriumaffinity in tris-based saline buffer containing 0.05% Tween-20 afterco-incubation of the antibody and human CCL17 for 48 hours at 4° C.

In some embodiments, the antibody binds human CCL17 with an affinityconstant (KD) of about 1×10⁻¹⁰ M or less, when the K_(D) is measuredusing solution equilibrium affinity in tris-based saline buffercontaining 0.05% Tween-20 after co-incubation of the antibody and humanCCL17 for 48 hours at 4° C.

In some embodiments, the antibody binds human CCL17 with the K_(D) ofabout 5×10⁻¹² M or less.

In another embodiment, the antibody of the invention specificallybinding human CCL17 binds Macaca fascicularis (cyno) CCL17 with anaffinity constant (K_(D)) of about 1×10⁻⁶ M or less, about 1×10⁻⁷ M orless, about 1×10⁻⁸ M or less, about 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M orless, about 1×10⁻¹¹ M or less or about 1×10⁻¹² M or less, when the K_(D)is measured using solution equilibrium affinity in tris-based salinebuffer containing 0.05% Tween-20 after co-incubation of the antibody andcyno CCL17 for 48 hours at 4° C. In some embodiments, the antibody ofthe invention binds Macaca fascicularis (cyno) CCL17 with the K_(D) ofabout 1×10⁻⁸M or less, when the K_(D) is measured using solutionequilibrium affinity in tris-based saline buffer containing 0.05%Tween-20 after co-incubation of the antibody and cyno CCL17 for 48 hoursat 4° C.

The affinity of an antibody to human CCL17 having the sequence of SEQ IDNO: 1 or cyno CCL17 having the sequence of SEQ ID NO: 2 can be measuredexperimentally using any suitable method. Such methods may utilizeProteon, Biacore or KinE×A instrumentation, such as ProteOn XPR36 orBiacore 3000, solution equilibrium affinity (SEA), ELISA or competitivebinding assays known to those skilled in the art. Exemplary methods arethose described in Example 3. The measured affinity of a particularantibody/CCL17 interaction can vary if measured under differentconditions (e.g., osmolarity, pH, buffer, detergent concentration).Thus, measurements of affinity and other binding parameters (e.g.,K_(D), K_(on), K_(off)) are preferably made with standardized conditionsand a standardized buffer, such as the buffer described herein. Skilledin the art will appreciate that the internal error for affinitymeasurements for example using solution equilibrium affinity, Biacore3000 or ProteOn (measured as standard deviation, SD) can typically bewithin 5-33% for measurements within the typical limits of detection.Therefore the term “about” reflects the typical standard deviation inthe assay. For example, the typical SD for a K_(D) of 1×10⁻⁹ M is up to±0.33×10⁻⁹M.

Another embodiment of the invention is an isolated antibody specificallybinding human CCL17, wherein the antibody inhibits CCL17 biologlca1act1vity.

“CCL17 biological activity” as used herein refers to any activityoccurring as a result of CCL17 binding to its receptor CCR4. Anexemplary CCL17 biological activity results in intracellular calciummobilization or chemotaxis of cells, for example CCRF-CEM cells (Tlymophblastoid cell line from patient with acute leukemia). Theantibodies of the invention can be tested for their ability to inhibitCCL17 biological activity using standard methods and those describedherein. For example, ability of. the antibodies of the invention toinhibit CCL17-dependent intracellular calcium mobilization can beassayed by measuring effect of the antibodies on CCL17-induced calciummobilization using fluorescent dyes such as Fluo-8 NW, Fluo-4 AM orFluo-3 AM. Ability of the antibodies of the invention to inhibitCCL17-induced chemotaxis can be measured by measuring migration ofCCRF-CEM cells through a semipermeable 5 μM filter in a two-chamberculture system, and measuring viability of cells migrated through thefilter, The antibodies of the invention may inhibit CCL17 biologicalactivity by about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Another embodiment of the invention is an antibody specifically bindingCCL17, wherein the antibody inhibits 10 ng/ml human CCL17-inducedcalcium mobilization in CCRF-CEM cells measured using Fluo-8 NW with anIC₅₀ value of about 1×10⁻⁷ M of less, about 1×10⁻⁸ M or less, or about1×10⁻⁹ M or less.

Another embodiment of the invention is an antibody specifically bindingCCL17, wherein the antibody comprises heavy chain complementaritydetermining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) and lightchain complementarity determining regions (LCDR) 1 (LCDR1), 2 (LCDR2)and 3 (LCDR3), wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 comprise the amino acid sequences of: SEQ ID NOs: 4,5, 71, 72, 73 and 74, respectively.

Antibodies comprising the HCDR and the LCDR sequences of SEQ ID NOs: 4,5, 11, 12, 73 and 74 bind human CCL17 with a K_(D) of 1×10⁻¹⁰ or less.

HCDR1: (SEQ ID NO: 4) SYWIG HCDR2: (SEQ ID NO: 5) IIDPSDSDTRYSPSFQGHCDR3 consensus sequence

(SEQ ID NO: 71) VGPADVWDX1FDY,

wherein

X₁ is s, A or T

x₂ is F, P, H or I;

x₃ is D, Y, W, T or V;

X₄ is I, F, S, T, Y, N, K or V; and

X₅ is K, A, Q, T or D.

LCDR2 consensus sequence:

(SEQ ID NO: 73) X1ASTRE,

wherein

X₁ is N, H, G, E, T or D.

LCDR3 consensus sequence

(SEQ ID NO: 74) QQX1X2X3X4PXsT;

wherein

x₁ is F, Y, T or H;

x₂ is Y, L, N or W;

x₃ is S, A, L, I, T, Q or H

x₄ is V, T, I, Y, L or D; and

X₅ is S, F, A or L.

In some embodiment, the HCDR1 comprises the sequence of SEQ ID NO: 4,the HCDR2 comprises the sequence of SEQ ID NO: 5 and the HCDR3 comprisesthe sequence of SEQ ID NOs: 6, 42, 43 or 44 in the antibody of theinvention specifically binding CCL17.

In some embodiments, the HCDR1 comprises the amino acid sequence of SEQID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5and the HCDR3 comprises the amino acid sequence of SEQ ID NOs: 6, 42 or43.

In some embodiments, the LCDR1 comprises the sequence of SEQ ID NOs: 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; the LCDR2 comprises thesequence of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 39, 40 or 41;and the LCDR3 comprises the sequence of SEQ ID NOs: 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37 or 38 in the antibody of the inventionspecifically binding CCL17.

In some embodiments, the LCDR1 comprises the amino acid sequence of SEQID NOs: 8, 9, 10, 13, 14, 15, 17 or 18, the LCDR2 comprises the aminoacid sequence of SEQ ID NOs: 20, 21, 22, 24, 25 and 26 and the LCDR3comprises the amino acid sequence of SEQ ID NOs: 28, 29, 30, 33, 34, 35,37 or 38.

In some embodiments, the antibody specifically binding CCL17 comprisesthe VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76.

VU consensus sequence (SEQ ID NO: 75)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVGPA DVWDX1FDYWGQGTLVTVSS

wherein

-   -   X₁ is s, A or T.

VL consensus sequence (SEQ ID NO: 76):DIVMTQSPDSLAVSLGERATINCKSSQSVLX1SX2X3NX4NX5LAWYQQKPGQPPLLIYX 6AS TRESGVPDRFSGSGSGTDFTLT IS SLQAEDVAVYYCQQX7X8X9X10 PX11 TFGQGTKVE IK;wherein

X₁ is L, Y, s or N;

X₂ is F, P, H or i;

X₃ is D, Y, W, T or V;

X₄ is I, F, S, T, Y, N, K or V;

X₅ is K, A, Q, T or D;

X₆ is N, H, G, E, T or D;

X₇ is F, Y, T or H;

X₈ is Y, L, N or W;

X₉ is S, A, L, I, T, Q or H;

X₁₀ is V, T, I , Y , L or D; and

X₁₁ is S, F, A or L.

In some embodiments, the antibody specifically binding CCL17 comprisesthe VH of SEQ ID NOs: 45, 46, 47 or 48.

In some embodiments, the antibody specifically binding CCL17 comprisesthe VL of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65 or 66.

In some embodiments, the antibody specifically binding CCL17 comprisesthe VH comprises the amino acid sequence of SEQ ID NOs: 45, 46 or 47.

In some embodiments, the antibody specifically binding CCL17 comprisesthe VL comprises the amino acid sequence of SEQ ID NOs: 50, 51, 52, 55,56, 57, 59, 60 or 62.

Another embodiment of the invention is an isolated antibody specificallybinding CCL17, wherein the antibody comprises the HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 sequences of

-   -   SEQ ID NOs: 4, 5, 6, 7, 19 and 27, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 20 and 28, respectively;    -   SEQ ID NOs: 4, 5, 6, 9, 21 and 29, respectively;    -   SEQ ID NOs: 4, 5, 6, 10, 22 and 30, respectively;    -   SEQ ID NOs: 4, 5, 6, 11, 23 and 31, respectively;    -   SEQ ID NOs: 4, 5, 6, 12, 24 and 32, respectively;    -   SEQ ID NOs: 4, 5, 6, 13, 21 and 33, respectively;    -   SEQ ID NOs: 4, 5, 6, 14, 20 and 34, respectively;    -   SEQ ID NOs: 4, 5, 6, 15, 25 and 35, respectively;    -   SEQ ID NOs: 4, 5, 6, 16, 21 and 36, respectively;    -   SEQ ID NOs: 4, 5, 6, 17, 25 and 37, respectively;    -   SEQ ID NOs: 4, 5, 6, 18, 26 and 38, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 39 and 26, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 24 and 28, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 22 and 28, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 40 and 28, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 26 and 28, respectively;    -   SEQ ID NOs: 4, 5, 6, 8, 41 and 28, respectively;    -   SEQ ID NOs: 4, 5, 42, 8, 24 and 28, respectively;    -   SEQ ID NOs: 4, 5, 43, 8, 24 and 28, respectively; or    -   SEQ ID NOs: 4, 5, 44, 8, 24 and 28, respectively.

Another embodiment of the invention is an isolated antibody specificallybinding CCL17, wherein the antibody comprises

the VH of SEQ ID NO: 45 and the VL of SEQ ID NOs: 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 58, 53, 60, 61, 62, 63, 64, 65 or 66;

the VH and the VL of SEQ ID NOs: 46 and 62, respectively;

the VH and the VL of SEQ ID NOs: 47 and 62, respectively; or

the VH and the VL of SEQ ID NOs: 43 and 62, respectively.

Another embodiment of the invention is an isolated antibody specificallybinding CCL17, wherein the antibody comprises

-   -   the VH of SEQ ID NO: 45 and the VL of SEQ ID NOs: 50, 51, 52,        55, 56, 57, 59 or 60;    -   the VH and the VL of SEQ ID NOs: 46 and 62, respectively; or    -   the VH and the VL of SEQ ID NOs: 47 and 62, respectively.

Another embodiment of the invention is an isolated antibody specificallybinding CCL17, wherein the antibody comprises the VH comprising theamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to the VH of SEQ ID NO: 46 and the VL comprising theamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to the VL of SEQ ID NO: 62.

Such exemplary antibodies are antibodies shown in Table 9.

Antibodies whose heavy chain CDR, light chain CDR, VH or VL amino acidsequences differ Insubstantially from those shown in Tables 3, 4, 6, 7and 9 are encompassed within the scope of the invention. Typically, thisinvolves one or more conservative amino acid substitutions with an aminoacid having similar charge, hydrophobic, or stereo chemicalcharacteristics in the antigen-binding site or in the framework, withoutadversely altering the properties of the antibody. Conservativesubstitutions may also be made to improve antibody properties, forexample stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 amino acid substitutions can be made to the VH or VLsequence. For example, a “conservative amino acid substitution” mayinvolve a substitution of a native amino acid residue with a nonnativeresidue such that there is little or no effect on the polarity or chargeof the amino acid residue at that position. Furthermore, any nativeresidue in the polypeptide may also be substituted with alanine, as hasbeen previously described for alanine scanning mutagenesis (MacLennan etal (1998) Act Physiol. Scand. Suppl. 643:55-67; Sasaki et al (1998) Adv.Biopsy's. 35:1-24). Desired amino acid substitutions can be determinedby those skilled in the art at the time such substitutions are desired.For example, amino acid substitutions can be used to identify importantresidues of the molecule sequence, or to increase or decrease theaffinity of the molecules described herein. The following eight groupscontain amino acids that are conservative amino acid substitutions forone another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)). Amino acid substitutions can be done forexample by PCR mutagenesis (U.S. Pat. No. 4,683,195). Libraries ofvariants can be generated using well known methods, for example usingrandom (NNK) or non-random codons, for example DVK codons, which encode11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)and screening the libraries for variants with desired properties.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy chain and one from a light chain, askilled artisan will recognize that alternative embodiments may comprisesingle heavy or light chain variable regions. The single variable regioncan be used to screen for variable domains capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to human CCL17 having the sequence of SEQ ID NO: 1. Thescreening may be accomplished by phage display screening methods usingfor example hierarchical dual combinatorial approach disclosed in Int.Pat. Publ. No. WO1932/01047. In this approach, an individual colonycontaining either a H or L chain clone is used to infect a completelibrary of clones encoding the other chain (L or H), and the resultingtwo-chain specific antigen-binding domain is selected in accordance withphage display techniques as described. Therefore, the individual VR andVL polypeptide chains are useful in identifying additional antibodiesspecifically binding human CCL17 having the sequence of SEQ ID NO: 1using the methods disclosed in Int. Pat. Publ. No. WO1992/01047.

Antibodies of the invention may be made using a variety of technologiesfor generating monoclonal antibodies. For example, the hybridoma methodof Kohler and Milstein, Nature 256:495, 1975 can be used. In thehybridoma method, a mouse or other host animal, such as a hamster, rator monkey, is immunized with human CCL17 and/or cyno CCL17 protein orfragments of these proteins, such as an extracellular portion of humanCCL17, followed by fusion of spleen cells from immunized animals withmyeloma cells using standard methods to form hybridoma cells (Gooding,Monoclonal Antibodies: Principles arid Practice, pp. 59-103 (AcademicPress, 1986)). Colonies arising from single immortalized hybridoma cellsare screened for production of antibodies with desired properties, suchas specificity of binding, cross-reactivity or lack thereof, andaffinity for the antigen.

Various host animals can be used to produce antibodies against humanCCL17. For example, Balb/c mice may be used to generate mouse anti-humanCCL17 antibodies. The antibodies made in Blab/c mice and other non-humananimals can be humanized using various technologies to generate morehuman-like sequences. Exemplary humanization techniques includingselection of human acceptor frameworks are known to skilled in the artand include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S.Pat. No. 6,318,749), Resurfacing (Palin, Mol Immunol 28:489-499, 1991),Specificity Determining Residues Resurfacing (U.S. Pat. Publ. No.2010/0261620), human-adaptation (or human framework adaptation) (U.S.Pat. Publ. No. US2009/0118127), Super humanization (U.S. Pat. No.7,709,226) and guided selection (Osborn et al., Methods 36:61-68, 2005;U.S. Pat. No. 5,565,332).

Humanized antibodies can be further optimized to improve theirselectivity or affinity to a desired antigen by incorporating alteredframework support residues to preserve binding affinity (back mutations)by techniques such as those disclosed as described in Int. Pat. Publ.No. WO1990/007861 and in Int. Pat. Publ. No. WO1992/22653.

Transgenic mice carrying human immunoglobulin loci in their genome canbe used to generate human antibodies against a target protein, and aredescribed in for example Int. Pat. Publ. No. WO1990/04036, U.S. Pat. No.6,150,584, Int. Pat. Publ. No. WO1999/45962, Int. Pat. Publ. No.WO2002/066630, Int. Pat. Publ. No. WO2002/43478, Loner et al., Nature368:856-9, 1994; Green et al., Nature Genet. 7:13-21, 1994; Green &Jakobovits Exp Med 188:483-95, 1998; Lonberg and Huszar Int Rev Immunol13:65-93, 1995; Bruggemann et al., Eur J Immunol 21:1323-1326, 1991;Fishwild et al., Nat Biotechnol 14:845-851, 1996; Mendez et al., NatGenet 15:146-156, 1997; Green, J Immunol Methods 231:11-23, 1999; Yanget al., Cancer Res 59:1236-1243, 1999; Brüggemann and Taussig Curr OpinBiotechnol 8:455-458, 1997; Int. Pat. Publ. No. WO2002/043478). Theendogenous immunoglobulin loci in such mice may be disrupted or deleted,and at least one complete or partial human immunoglobulin locus may beinserted into the mouse genome using homologous or non-homologousrecombination, using transchromosomes, or using minigenes. Companiessuch as Regeneron, Harbour Antibodies, Open Monoclonal Technology, Inc.(OMT), KyMab, Trianni and Abiexis can be engaged to provide humanantibodies directed against a selected antigen using technology asdescribed above.

Human antibodies can be selected from a phage display library, where thephage is engineered to express human immunoglobulins or portions thereofsuch as Fabs, single chain antibodies (scFv), or unpaired or pairedantibody variable regions (Knappik et al., J Mol Biol 296:57-86, 2000;Krebs et al., J Immunol Meth 254:67-84, 2001; Vaughan et al., NatureBiotechnology 14:309-314, 1996; Sheets et al., PITAS(USA) 95:6157-6162,1998; Hoogenbcom and Winter, J Mol Biol 227:381, 1991; Marks et al., JMol Biol 222:581, 1991). The antibodies of the invention may be isolatedfor example from phage display library expressing antibody heavy andlight chain variable regions as fusion proteins with bacteriophage pIXcoat protein as described in Shi et al., J Mol Biol 397:385-96, 2010 andInt. Pat. Publ. No. WO2009/085462). The antibody libraries are screenedfor binding to human CCL17 extracellular domain and the obtainedpositive clones are further characterized, the Fabs isolated from theclone lysates, and expressed as full length IgGs. Such phage displaymethods for isolating human antibodies are established in the art. Seefor example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 toLadner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.;U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S.Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and6,593,081 to Griffiths et al.

Preparation of immunogenic antigens and monoclonal antibody productioncan be performed using any suitable technique, such as recombinantprotein production. The immunogenic antigens can be administered to ananimal in the form of purified protein, or protein mixtures includingwhole cells or cell or tissue extracts, or the antigen can be formed denovo in the animal's body from nucleic acids encoding said antigen or aportion thereof.

The antibodies of the invention may be human or humanized.

The antibodies of the invention may be synthetic or recombinant.

The antibodies of the invention may be of IgA, IgD, IgE, IgC or IgMtype. The antibodies of the invention may be of IgG1, IgG2, IgG3, IgG4isotype.

Immune effector properties of the antibodies of the invention may beenhanced or silenced through Fc modifications by techniques known tothose skilled in the art. For example, Fc effector functions such as Clqbinding, complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. can be modulated bymodifying residues in the Fc responsible for these activities.Pharmacokinetic properties may also be enhanced by mutating residues inthe Fc domain that extend antibody half-life. Exemplary Fc modificationsare IgG4 S228P/L234A/L-235A, IgG2 M252Y/S254T/T256E (Dall'Acqua et al.,J Biol Chem 281:23514-24, 2006; or IgG2 V234A/G237A/P238S,V234A/G237A/H268Q, H268A/V309L/A330S/P331 orV234A/G237A/P233S/H268A/V309L/A330S/P331S on IgG2 (Intl. Pat. Publ. No.W02011/066501), or those described in US. Pat. No. 6,737,056 (numberingaccording to the EU numbering).

In some embodiments, the antibody specifically binding human CCL17comprises a substitution in an Fc region.

In some embodiments, the substitution comprises V234A, G237A, P238S,H268A, V309L, A330S or P331S substitution on IgG2, or S228P, L234A orL235A substitution on IgG4, wherein residue numbering is according tothe EU Index.

Additionally, antibodies of the invention can be post-translationallymodified by processes such as glycosylation, isomerization,deglycosylation or non-naturally occurring covalent modification such asthe addition of polyethylene glycol moieties (pegylation) andlipidation. Such modifications may occur in vivo or in vitro. Forexample, the antibodies of the invention can be conjugated topolyethylene glycol (PEGylated) to improve their pharmacokineticprofiles. Conjugation can be carried out by techniques known to thoseskilled in the art. Conjugation of therapeutic antibodies with PEG hasbeen shown to enhance pharmacodynamics while not interfering withfunction (Knight et al., Plateles 15:409-418, 2004; Leong et al.,Cytokine 16:106-119, 2001; Yang et al., Protein Eng 16:761-770, 2003).

Antibodies or fragments thereof of the invention modified to improvestability, selectivity, cross-reactivity, affinity, immunogenicity orother desirable biological or biophysical property are within the scopeof the invention. Stability of an antibody is influenced by a number offactors, including (1) core packing of individual domains that affectstheir intrinsic stability, (2) protein/protein interface .interactionsthat have impact upon the HC and LC pairing, (3) burial of polar andcharged residues, (4) H-bonding network for polar arid charged residues;and (5) surface charge and polar residue distribution among other intra-and inter-molecular forces (Worn and Pluckthun, J Mol Biol 305:989-1010,2001). Potential structure destabilizing residues may be identifiedbased upon the crystal structure of the antibody or by molecularmodeling in certain cases, and the effect of the residues on antibodystability can be tested by generating and evaluating variants harboringmutations in the identified residues. One of the ways to increaseantibody stability is to raise the thermal transition midpoint (T_(m))as measured by differential scanning calorimetry (DSC) . In general, theprotein T_(m) is correlated with its stability and inversely correlatedwith its susceptibility to unfolding and denaturation in solution andthe degradation processes that depend on the tendency of the protein tounfold (Remmele et al., Pharm Res 15:200-208, 1997). A number of studieshave found correlation between the ranking of the physical stability offormulations measured as thermal stability by DSC and physical stabilitymeasured by other methods (Bedu-Addo et al., Pharm Res 21:1353-1361,2004; Gupta and Kaisheva, AAPS PharSci, 5E8, 2003; Maa and Hsu, Int JPharm 140:155-168, 1996; Remmele et al., Pharm Res 15:200-208, 1997;Zhang et al., J Pharm Sci 93:3076-3089, 2004). Formulation studiessuggest that a Fab Tm has implication for long-term physical stabilityof a corresponding mAb. Differences in amino acids in either frameworkor within the CDRs could have significant effects on the thermalstability of the Fab domain (Yasui et al., FEBS Lett 353:143-146, 1994).

CCL17 antibodies of the invention can be engineered into bispecificantibodies which are also encompassed within the scope of the invention.The VL and/or the VH regions of the antibodies of the invention can beengineered using published methods into single chain bispecificantibodies as structures such as TandAb® designs (Int. Pat. Publ. No.WO1999/57150; U.S. Pat. Publ. No. US2011/0206672) or into bispecificscFVs as structures such as those disclosed in U.S. Pat. No. 5,869,620;Int. Pat. Publ. No. WO1995/15388, int. Pat. Publ. No. WO1997/14719 orInt. Pat. Publ. No WO2011/036460.

The VL and/or the VH regions of the antibodies of the invention can beengineered into bispecific full length antibodies, where each antibodyarm binds a distinct antigen or epitope. Such bispecific antibodies aretypically made by modulating the CH3 interactions between the twoantibodies heavy chains to form bispecific antibodies using technologiessuch as those described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No.WO2004/111233; U.S. Pat. Publ. No. US2010/0015133; U.S. Pat. Publ. No.US2007/0287170; Int. Pat. Publ. No. WO2008/119353; U.S. Pat. Publ. No.US2009/0182127; U.S. Pat. Publ. No. US2010/0286374; U.S. Pat. Publ. No.US2011/0123532; Int. Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No.WO2011/143545; or U.S. Pat. Publ. No. US2012/0149876. Additionalbispecific structures into which the VL and/or the VH regions of theantibodies of the invention can be incorporated are for example DualVariable Domain Immunoglobulins (Int. Pat. Publ. No. WO2009/134776), orstructures that include various dimerization domains to connect the twoantibody arms with different specificity, such as leucine zipper orcollagen dimerization domains (Int. Pat. Publ. No. WO2012/022811, U.S.Pat. Nos. 5,932,448; 6,833,441).

Another embodiment of the invention is an isolated polynucleotideencoding any of the antibody heavy chain variable regions or theantibody light chain variable regions of the invention. Certainexemplary polynucleotides are disclosed herein, however, otherpolynucleotides which, given the degeneracy of the genetic code or codonpreferences in a given expression system, encode the antibodies of theinvention are also within the scope of the invention. The polynucleotidesequences encoding a VH or a VL or a fragment thereof of the antibody ofthe invention can be operably linked to one or more regulatory elements,such as a promoter and enhancer, that allow expression of the nucleotidesequence in the intended host cell. The polynucleotide may be a cDNA.

Another embodiment of the invention is a vector comprising thepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of thepolynucleotide of the invention into a given organism or geneticbackground by any means. For example, polynucleotides encoding light andheavy chain variable regions of the antibodies of the invention,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include signal sequences, promoters (e.g.naturally associated or heterologous promoters), enhancer elements, andtranscription termination sequences, and are chosen to be compatiblewith the host cell chosen to express the antibody. Once the vector hasbeen incorporated into the appropriate host, the host, is maintainedunder conditions suitable for high level expression of the proteinsencoded by the incorporated polynucleotides.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part, of the hostchromosomal DNA. Commonly, expression vectors contain selection markerssuch as ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance to permitdetection of those cells transformed with the desired DMA sequences.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, exemplary promoters include lad, lacZ,T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell,exemplary promoters include light and/or heavy chain immunoglobulin genepromoter and enhancer elements; cytomegalovirus immediate earlypromoter; herpes simplex virus thymidine kinase promoter; early and lateSV40 promoters; promoter present in long terminal repeats from aretrovirus; mouse metailothionein-I promoter; and various art-knowntissue specific promoters. For expression in a yeast cell, an exemplarypromoter is constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBiuescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Another embodiment of the invention is a host cell comprising the vectorof the invention. The term “host cell” refers to a cell into which avector has been introduced. It is understood that the term host cell isintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not be identical to the parent cell, butare still included within the scope of the term “host cell” as usedherein. Such host cells may be eukaryotic cells, prokaryotic cells,plant cells or archeal cells.

Escherichia coli, bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species are examples of prokaryotic host cells. Othermicrobes, such as yeast, are also useful for expression. Saccharomyces(e.g., S. cerevisiae) arid Pichia are examples of suitable yeast hostcells. Exemplary eukaryotic cells may be of mammalian, insect, avian orother animal origins. Mammalian eukaryotic cells include immortalizedcell lines such as hybridomas or myeloma cell lines such as SP2/0(American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NS0(European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,ECACC No. 85110503), FC (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murinecell lines. An exemplary human myeloma cell line is U266 (ATTCCRL-TIB-196). Other useful cell lines include those derived from ChineseHamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologies,Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.

Another embodiment of the invention is a method of producing an antibodyof the invention comprising culturing the host cell of the invention andrecovering the antibody produced by the host cell. Methods of makingantibodies and purifying them are well known in the art. Oncesynthesized (either chemically or recombinantly), the whole antibodies,their dimers, individual light and heavy chains, or other antibodyfragments such as VH or VL, can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, high performance liquidchromatography (HPLC) purification, gel electrophoresis, and the like(see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,(1982)). A subject antibody can be substantially pure, e.g., at leastabout 80% to 85% pure, at least about 85% to 90% pure, at least about90% to 95% pure, or at least about 98% to 99%, or more, pure, e.g., freefrom contaminants such as cell debris, macromolecules other than asubject antibody, etc.

The polynucleotides encoding certain VH or VL sequences of the inventionare incorporated into vectors using standard molecular biology methods.Host cell transformation, culture, antibody expression and purificationare done using well known methods.

Methods of Treatment

Antibodies specifically binding human CCL17 may be suitable for treatingor preventing a spectrum of CCL17-mediated conditions.

The term “CCL17-mediated condition” as used herein encompasses alldiseases and medical conditions in which CCL17 plays a role, whetherdirectly or indirectly, in the disease or medical condition, includingthe causation, development, progress, persistence or pathology of thedisease or condition.

The term “CCL17-mediated inflammatory condition” as used herein refersto an inflammatory condition resulting at least partially from CCL17biological activity. Exemplary CCL17-mediated inflammatory conditionsare asthma and allergies.

The methods of the invention may be used to treat an animal patientbelonging to any classification. Examples of such animals includemammals such as humans, rodents, dogs, cats and farm animals. Forexample, the antibodies of the invention are useful in the prophylaxisand treatment of CCL17-mediated conditions, such as asthma andrespiratory allergic diseases such as allergic asthma, allergicrhinitis, chronic obstructive pulmonary disease (COPD), idiopathicpulmonary fibrosis (IPF), hypersensitivity lung diseases and the like,allergic diseases such as systemic anaphylaxis or hypersensitivityresponses, drug allergies, allergic bronchopulmonary aspergillosis(ABPA), insect, sting allergies and food allergies, inflammatory boweldiseases such as Crohn's disease, ulcerative colitis, ileitis andenteritis, vaginitis, psoriasis and inflammatory dermatoses such asdermatitis, eczema, atopic dermatitis, allergic contact dermatitis,urticaria and pruritus, vasculitis, spondyloarthropathies, scleroderma,autoimmune diseases, such as arthritis (including rheumatoid andpsoriatic), multiple sclerosis, systemic lupus erythematosus, type Idiabetes, glomerulonephritis, and the like, graft rejection (includingallograft rejection and graft-v-host disease), and other diseases inwhich undesired inflammatory responses are to be inhibited, such asatherosclerosis, myositis, T-cell mediated neurodegenerative diseases,multiple sclerosis, encephalitis, meningitis, hepatitis, nephritis,sepsis, sarcoidosis, allergic conjunctivitis, otitis, Castleman'sdisease, sinusitis, LPS-induced endotoxic shock, Behcet's syndrome andgout.

The antibodies of the invention and are also useful in the preparationof a medicament for such treatment, wherein the medicament is preparedfor administration in dosages defined herein.

The methods and uses of the present may be intended for use in animalsand patients that have, or are at risk for developing any disease orcondition, associated with CCL17 expression or biological activity or inwhich CCL17 plays a biological role.

By not wishing to be bound by any theory, the antibodies of theinvention may provide their efficacious effect in various inflammatorydiseases by direct inhibition of Th2 cell recruitment and thereforesimultaneous inhibition of multiple Th2 cytokines. The antibodies of theinvention may provide an improved safety profile in comparison toanti-CCR4 antibodies by selectively blocking CCL17 only. The antibodieswill not interact with platelets, which express CCR4. In addition, theantibodies will not block the beneficial innate immune effects of CCL22on CCR4 (Matsukawa et al., I. Immunol 164:5382-8, 2000).

“Inflammatory condition” as used herein refers to acute or chroniclocalized or systemic responses to harmful stimuli, such as pathogens,damaged cells, physical injury or irritants, that are mediated in partby the activity of cytokines, chemokines, or inflammatory cells (e.g.,neutrophils, monocytes, lymphocytes, macrophages, mast cells, dendriticcells, neutrophils) and is characterized in most instances by pain,redness, swelling, and impairment of tissue function.

Inflammatory pulmonary condition is an example of a CCL17-mediatedinflammatory condition. Exemplary inflammatory pulmonary conditionsinclude infection-induced pulmonary conditions including thoseassociated with viral, bacterial, fungal, parasite or prion infections;allergen-induced pulmonary conditions; pollutant-induced pulmonaryconditions such as asbestosis, silicosis, or berylliosis; gastricaspiration-induced pulmonary conditions, immune dysregulation,inflammatory conditions with genetic predisposition such as cysticfibrosis, and physical trauma-induced pulmonary conditions, such asventilator injury. These inflammatory conditions also include asthma,emphysema, bronchitis, chronic obstructive pulmonary disease (COPD),sarcoidosis, histiocytosis, lymphangiomyomatosis/acute lung injury,acute respiratory distress syndrome, chronic lung disease,bronchopulmonary dysplasia, community-acquired pneumonia, nosocomialpneumonia, ventilator-associated pneumonia, sepsis, viral pneumonia,influenza infection, parainfluenza infection, rotavirus infection, humanmetapneumovirus infection, respiratory syncitial virus infection andAspergillus or other fungal infections. Exemplary infection-associatedinflammatory diseases may include viral or bacterial pneumonia,including severe pneumonia, cystic fibrosis, bronchitis, airwayexacerbations and acute respiratory distress syndrome (ARDS). Suchinfection-associated conditions may involve multiple infections such asa primary viral infection and a secondary bacterial infection.

Asthma is an inflammatory disease of the lung that is characterized byairway hyperresponsiveness (“AHR”), bronchoconstriction, wheezing,eosinophilic or neutrophilic inflammation, mucus hypersecretion,subepithelial fibrosis, and elevated IgE levels. Patients with asthmaexperience “exacerbations”, a worsening of symptoms, most commonly dueto microbial infections of the respiratory tract (e.g. rhinovirus,influenza virus, Haemophilus influenza, etc.). Asthmatic attacks can betriggered by environmental factors (e.g. ascarids, insects, animals(e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs andbirds), fungi, air pollutants (e.g., tobacco smoke), irritant gases,fumes, vapors, aerosols, chemicals, pollen, exercise, or cold air. Apartfrom asthma, several chronic inflammatory diseases affecting the lungare characterized by neutrophil infiltration to the airways, for examplechronic obstructive pulmonary disease (COPD), bacterial pneumonia andcystic fibrosis (Linden et al., Eur Respir J 15:973-7, 2000; Rahman etal., Clin Immunol 115:268-76, 2005), and diseases such as COPD, allergicrhinitis, and cystic fibrosis are characterized by airwayhyperresponsiveness (Fahy and O'Byrne Am J Respir Crit Care Med163:822-3, 2001).

In allergic asthma, the presence of high levels of allergen-specific IgEis a reflection of an aberrant Th2 cell immune response to commonlyinhaled environmental allergens. Allergens are presented to T cells bydendritic cells (DCs) that, continuously sample incoming foreignantigens. Upon proper activation by DCs, allergen-specific lymphocytesthat are present in diseased airways produce Th2 cytokines interleukin(IL)-4, IL-5 and IL-13 that furthermore control leukocyte extravasation,goblet cell hyperplasia and bronchial hyper-reactivity (BHR). CCL17produced by DCs induce the selective migration of Th2 cells but not Th1cells through triggering CCR4. In murine models of asthma, thattreatment with anti-CCL17 antibodies reduced the number of CD4+ T cellsand eosincohils in bronchoaiveolar lavage (BAL) fluid, the production ofTh2 cytokines and airway hyper-responsiveness after allergen challenge,suggesting that CCL17 neutralization is a feasible strategy forinhibiting allergic inflammation in humans.

Commonly used animal models for asthma and airway inflammation includethe ovalbumin challenge model, methacholine sensitization models andsensitization with Aspergillus fumigatus (Hessel et al., Eur J Pharmacol293:401-12, 1995). Inhibition of cytokine and chemokine production fromcultured human bronchial epithelial cells, bronchial fibroblasts orairway smooth muscle cells can also be used as in vitro models. Theadministration of antibodies of the invention to any of these models canbe used to evaluate the efficacy to ameliorate symptoms and alter thecourse of asthma, airway inflamination, COPD and the like.

Atopic dermatitis is an example of a CCL17-mediated inflammatorycondition.

One aspect of the invention is a method of treating a CCL17-mediateddisease, comprising administering to a subject in need thereof theantibody of the invention for a time sufficient to treat theCCL17-mediated disease.

In some embodiments, the CCL17-mediated disease is an inflammatorydisease.

In some embodiments, the inflammatory disease is asthma, ulcerativecolitis (UC), atopic dermatitis (AD) or idiopathic pulmonary fibrosis(IPF).

One embodiment of the invention is a method of treating asthma,comprising administering to a subject an antibody of the invention for atime sufficient to treat asthma.

Administration/Pharmaceutical Compositions

The invention provides for pharmaceutical compositions comprising theantibodies specifically binding CCL17 of the invention of the inventionand a pharmaceutically acceptable carrier. For therapeutic use, theantibodies specifically binding CCL17 of the invention may be preparedas pharmaceutical compositions containing an effective amount of thedomain, molecule or antibody as an active ingredient in apharmaceutically acceptable carrier. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the active compoundis administered. Such vehicles may be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.For example, 0.4% saline and 0.3% glycine can be used. These solutionsare sterile and generally free of particulate matter. They may besterilized by conventional, well-known sterilization techniques (e.g.,filtration). The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, stabilizing, thickening,lubricating and coloring agents, etc. The concentration of the moleculesor antibodies of the invention in such pharmaceutical formulation mayvary widely, i.e., from less than about 0.3%, usually at or at leastabout 1% to as much as 15 or 20% by weight and will be selectedprimarily based on required dose, fluid volumes, viscosities, etc.,according to the particular mode of administration selected. Suitablevehicles and formulations, inclusive of. other human proteins, e.g.,human serum albumin, are described, for example, in e.g. Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, Troy, D. B. ed.,Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5,Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the antibodiesspecifically binding CCL17 of the invention may be any suitable routethat delivers the agent to the host/such as parenteral administration,e.g., intradermal, intramuscular, intraperitoneal, intravenous orsubcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal,rectal); using a formulation in a tablet, capsule, solution, powder,gel, particle; and contained in a syringe, an implanted device, osmoticpump, cartridge, micropump; or other means appreciated by the skilledartisan, as well known in the art. Site specific administration may beachieved by for example intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intracardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravascular, intravesical,intralesional, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal delivery.

Thus, a pharmaceutical composition of the invention for intramuscularinjection may be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg/kg, e.g. about 50 ng to about 30mg/kg or more preferably, about 5 mg to about 25 mg/kg, of theantibodies specifically binding CCL17 of the invention.

The dose given to a patient having a CCL17-mediated condition issufficient to alleviate symptoms or treat the CCL17-mediated condition(“therapeutically effective amount”) and is sometimes 0.1 to 10 mg/kgbody weight, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but mayeven higher, for example 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100mg/kg. A fixed unit dose may also be given, for example, 50, 100, 200,500 or 1000 mg, or the dose may be based on the patient's surface area,e.g., 400, 300, 250, 200, or 10 mg/m². Usually between 1 and 8 doses,(e.g., 1, 2, 3, 4, 5, 6, 7 or 8) are administered, but 10, 12, 20 ormore doses may be given. Administration of the antibodies specificallybinding CCL17 of the invention may be repeated after one day, two days,three days, four days, five days, six days, one week, two weeks, threeweeks, one month, five weeks, six weeks, seven weeks, two months, threemonths, four months, five months, six months or longer. Repeated coursesof treatment are also possible, as is chronic administration. Therepeated administration may be at the same dose or at a different dose.

The dosage of the CCL17 antibodies of the invention that will beeffective in the treatment of. inflammatory diseases such as asthma canbe determined by administering the CCL17 antibodies to relevant animalmodels well known in the art and as described herein.

In vitro assays can optionally be employed to help identify optimaldosage ranges. Selection of a particular effective dose can bedetermined (e.g., via clinical trials) by those skilled in the art basedupon the consideration of several factors. Such factors include thedisease to be treated or prevented, the symptoms involved, the patient'sbody mass, the patient's immune status and other factors known by theskilled artisan. The precise dose to be employed in the formulation willalso depend on the route of administration, and the severity of disease,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.The antibodies of the invention may be tested for their efficacy andeffective dosage using any of the models described herein.

For example, a pharmaceutical composition comprising the antibodiesspecifically binding CCL17 of the invention for intravenous infusion maybe made up to contain about 200 ml of sterile Ringer's solution, andabout 8 mg to about 2400 mg, about 400 mg to about 1600 mg, or about 400mg to about 500 mg of the antibodies specifically binding CCL17 of theinvention for administration to a 80 kg patient. Methods for preparingparenteraliy administrable compositions are well known and are describedin more detail in, for example, “Remington's Pharmaceutical Science”,15th ed., Mack Publishing Company, Easton, Pa.

The antibodies specifically binding CCL17 of the invention may belyophilized for storage and reconstituted in a suitable carrier prior touse. This technique has been shown to be effective with conventionalprotein preparations and art-known lyophilization and reconstitutiontechniques can be employed.

The antibodies specifically binding CCL17 of the invention may beadministered in combination with a second therapeutic agentsimultaneously, sequentially or separately.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

EXAMPLE 1 Generation of CCL17 Neutralizing Antibodies

Human CCL17 binding Fabs were selected from de novo pIX phage displaylibraries described in Shi et al., J. Mol. Biol. 397:385-396, 2010; Int.Pat. Publ. No. WO2009/085462; U.S. Pat. Publ. No. US2010/0021477; U.S.Pat. Publ. No. US2012/0105795. Briefly, the libraries were generated bydiversifying human scaffolds where germline VH genes IGHV1-69*01,IGHV3-23*01, and IGKV5-51*01 were recombined with the human IGHJ-4minigene via the H3 loop, and human germline VL kappa genes 012(IGKV1-39*01), L6 (IGKV3-11*01) , A27 (IGKV3-20*01), and B3 (IGKV4-1*01)were recombined with the IGKJ-1 minigene to assemble complete VH and VLdomains. The positions in the heavy and light chain variable regionsaround H1, H2, L1, L2 and L3 loops corresponding to positions identifiedto be frequently in contact with protein and peptide antigens werechosen for diversification. Sequence diversity at selected positions waslimited to residues occurring at each position in the IGHV or IGLVgermline gene families of the respective IGHV or XGLV genes. Diversityat the H3 loop was generated by utilizing short to mid-sized syntheticloops of lengths 7-14 amino acids. The amino acid distribution at H3 wasdesigned to mimic the observed variation of amino acids in humanantibodies. Library design is detailed in Shi et al., J Mol Biol397:385-96, 2010. The scaffolds utilized to generate libraries werenamed according to their human VH and VL germline gene origin. The threeheavy chain libraries were combined with the four germline light chainsor germline light chain libraries to generate 24 unique VH:VLcombinations for screening. All 24 VH:VL library combinations wereutilized in phage panning experiments against human CCL17.

The libraries were panned using human CCL17 of SEQ ID NO: 1. Briefly,human CCL17 was biotinylated using standard methods and the biotinylatedhuman CCL17 (Bt-huCCL17) was captured on streptavidin-coated magneticbeads (Dynal, M280), and the Fab-pIX phage libraries were added to thebeads. Bt-huCCL17 concentrations used were 100 nM for rounds 1 and 2 and10 mM for rounds 3 and 4). Screening was done by ELISA for Fab bindingto human CCL17 protein. For panning, the bt-huCCL17 coated magneticbeads were washed and blocked with PBST-M (BPS with 0.05% Tween-20 and3% non-fat dry milk), Blocked phage from the libraries were added to thebeads and rotated at room temperature for round 1. Beads were washed andthen incubated in a culture of log phase E. coli, (MC1061F′) cells andthe infected E. coli were grown on LB agar plates overnight at 37° C.The next morning cultures were scraped from the plates in 2 mL 2×YT (20%glycerol) per plate, 50 μL of bacterial suspension was added to 50 mL2×YT (Carb) and grown at 37° C. shaking for up to 2 hours. Helper phagewas added to mid-log phase cultures and the cultures were incubated at37° C. for 30 minutes. Kanamycin and IPTG were added to each culture tofinal concentrations of 35 λg/mL and 1 mM, respectively, and grownovernight at 30° C. shaking. The amplified phage from the bacterialmedia was precipitated using PEG/NaCl and re-suspended in 1 mL PBS. 200μL was used for the next round of panning.

After three rounds of panning, phagemid DMA was isolated from theinfected MC1061F′ cells and digested with restriction enzymes to removethe sequence encoding pIX, and the linearized plasmid DMA was excisedand purified from agarose gels. This DNA was then self-ligated with T4DNA ligase. The ligated DNA was electroporated into MC1061F′ cells andplated onto LB (Carb/Glucose) agar plates. Colonies from thiselectroporation were picked for the ELISA screen and assessment of Fabexpression. The Fabs contain an in-frame His tag at the C-terminus ofthe heavy chain. From the initial screen, 24 Fabs were partiallypurified via the C-terminal His tag using standard methods andcharacterized further.

The Fabs were characterized for their binding to human CCL17 (SEQ ID NO:1), cyno CCL17 (SEQ ID NO: 2) and cyno CCL22 (SEQ ID NO: 3) in an ELISAassay. Briefly, Maxisorp 96 well plates were coated with 1 μg/ml Goatanti-human Kappa (Southern Biotech). Semi-purified Fab was added to eachplate. Biotinylated huCCL17, cCCL17 or cCCL22 was added to eachFab-captured well. Proteins bound to the captured Fabs were detectedusing Streptavidin:HRP. Five Fabs (F21, F24, F34, F43 and F44) thatbound to both human and cyno CCL17 but not cync CCL22 were selected foraffinity maturation.

EXAMPLE 2 Affinity Maturation of Anti-CCL17 Antibodies

Five Fabs were selected for affinity maturation based on their initialcharacterization profile. The Fabs were affinity-matured by diversifyingthe light chains using the in-line maturation technology described inShi et al. (Shi et al., J Mol Biol 397:385-396, 2010) and keeping theheavy chain invariant. The heavy chain in the Fabs was either VH3-23 orVH5-51. F24 affinity maturation libraries produced improved CCL17binders.

Briefly, F24 was affinity-matured using the B3 light chain library. TheB3 library diversification scheme is shown in Table 2. The positions areindicated as Kabat numbering.

TABLE 2 Residue Germline Library position (Kabat) residue compositions 27d Y S, Y, H, F, or A 30 K K, T, N, or E 32 Y Y, F, H, N, W, D, A, orS 50 W Y, W, S, R, D, Y, or A 91 Y Y, S, H, or A 92 Y Y, N, D, S, H, I,F, or K 93 S S, N, T, D, G, H, or R 94 T T, Y, L, V. F, A, or S 96 L W,Y, F, L, I, or R

The VH regions of the Fabs were cloned into the LC library phagemidresulting in a complete re-diversification of the LC for each Fab. VHregions were isolated by restriction digestion of DNA minipreps usingNcoI and ApaI. The VH regions were gel isolated and ligated intosimilarly digested LC library DNA. Ligations were purified andtransformed into MC1061F′ cells. Cells were grown in 2×YT (Carb) untillog phase growth (OD_(600nm)≈0.6) was achieved. Helper phage was addedand the cultures were incubated at 37° C. for 30 minutes. Kanamycin andIPTG were added to each culture to final concentrations of 35 μg/mL and1 mM, respectively, and grown overnight at 30° C. shaking. The phagefrom the bacterial media was precipitated using PEG/NaCl andre-suspended in PBS.

For affinity maturation panning, Bt-CCL17 was captured on 50 μl ofSA-coated magnetic beads. Antigen concentrations were 100 nM for round1, 10 nM for round 2, and 10 nM for round 3. Beads were subjected to 6washes with PBST and one wash with PBS, followed by E. coli infection asdescribed above. Isolation of Fab expression plasmids and expression ofFabs was done as described.

Affinity-matured Fabs were screened in an ELISA assay for binding tohuCCL17 (SEQ ID NO: 1), cCCL17 (SEQ ID NO: 2) and eCCL22 (SEQ ID NO: 3)as described above for binding to huCCL17. Identified clones weresequenced, converted to full IgG1 antibodies and their binding tohuCCL17, cCCL17 and cCCL22 was confirmed using MSD-SEA.

CDR sequences of the parent and affinity-matured antibodies are shown inTable 3 and Table 4 for heavy chain and light chain CDRs, respectively.

TABLE 3 HDCR1 HCDR2 HCDR3 SEQ ID SEQ ID SEQ ID mAb ID sequence NO:sequence NO: sequence NO: C17F24 SYWIG 4 IIDPSDSDTRYSPSFQG 5VGPADVWDSFDY 6 (parent) C17B234 SYWIG 4 IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY6 C17B235 SYWIG 4 IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6 C17B236 SYWIG 4IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6 C17B237 SYWIG 4 IIDPSDSDTRYSPSFQG 5VGPADVWDSFDY 6 C17B238 SYWIG 4 IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6C17B239 SYWIG 4 IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6 C17B240 SYWIG 4IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6 C17B241 SYWIG 4 IIDPSDSDTRYSPSFQG 5VGPADVWDSFDY 6 C17B242 SYWIG 4 IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6C17B243 SYWIG 4 IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6 C17B244 SYWIG 4IIDPSDSDTRYSPSFQG 5 VGPADVWDSFDY 6

TABLE 4 LCDR1 LCDR2 LCDR3 SEQ SEQ SEQ ID ID ID mAb ID sequence NO:sequence NO: sequence NO: C17F24 KSSQSVLYSS 7 WASTRES 19 QQYYSTPLT 27(parent) NNKNYLA C17B234 KSSQSVLLSF 8 NASTRES 20 QQFYSVPST 28 DNINKLAC17B235 KSSQSVLYSF 9 HASTRES 21 QQFYATPFT 29 YNFNALA C17B236 KSSQSVLLSP10 GASTRES 22 QQYYLIPST 30 WNSNQLA C17B237 KSSQSVLTSY 11 LASTRES 23QQYLSPPST 31 NNSNYLA C17B238 KSSQSVLISA 12 DASTRES 24 QQYQFIPFT 32FNQNPLA C17B239 KSSQSVLSSF 13 HASTRES 21 QQYLIYPST 33 TNTNTLA C17B240KSSQSVLYSH 14 NASTRES 20 QQYYTLPAT 34 VNYNALA C17B241 KSSQSVLNSF 15EASTRES 25 QQTNSIPLT 35 TNNNALA C17B242 KSSQSVLFSH 16 HASTRES 21QQYYAVPQT 36 DNLNTLA C17B243 KSSQSVLNSF 17 EASTRES 25 QQHWQTPLT 37DNKNDLA C17B244 KSSQSVLSSI 18 TASTRES 26 QQYYHDPFT 38 TNVNDLA

EXAMPLE 3 Binding of Affinity-Matured Anti-CCL17 Antibodies to Human andCyno CCL17

Antibodies were assessed for their binding to human CCL17 and cyno CCL17using solution equilibrium affinity (SEA). The procedure for theseexperiments was similar to that used by Haenel et al (Haenel et al.,Anal Biochem 339:182-184, 2005). To prepare the antigen-antibodycomplexes, human CCL17 or cyno CCL17 was serially diluted in Tris-BasedSaline buffer containing 0.05% Tween-20, TBST, (Thermo Scientific) at a1:6 ratio starting at a concentration of 2,000,000 μM in 96-deep wellpolypropylene plates. Equal volumes of the anti-hCCL17 mAbs at 40 pM or200 pM were added to each chemokine dilution to obtain mixturescontaining the serial dilution of chemokines starting at a finalconcentration of 1 pM and a constant concentration (20 pM or 100 pM) ofanti-CCL17 antibody. The mixtures were prepared in duplicate andincubated at 4° C. for 48 hours to reach equilibrium. The free antibodywas detected using a SECTOR Imager 6000 (Meso Scale Discovery)instrument. The resulting binding curves were fitted to obtain thedissociation equilibrium constant (K_(D)) using the GraphPad Prismsoftware (v 5.01) using a 1:1 binding model to perform nonlinearleast-square regression analysis of the data. Table 5 shows affinitiesof the antibodies to human and cyno CCL17. The affinities ranged fromabout 2 pM to about 700 pM for human CCL17 and from about 200 pM toabout 9500 pM to cyno CCL17. The generated antibodies bound human CCL17with about 2- to about 150 fold higher affinities when compared tobinding to cyno CCL17.

TABLE 5 Affinity (pM) Fold binding mAb ID human CCL17 cyno CCL17human/cyno CCL17 C17F24 1000 ND* (parent) C17B234 2 230 115.0 C17B235 729497 131.9 C17B236 39 297 7.6 C17B237 657 3066 4.7 C17B238 115 1456 12.7C17B239 92 630 6.8 C17B240 83 4596 55.4 C17B241 50 583 11.7 C17B242 167384 2.3 C17B243 28 677 24.2 C17B244 33 565 17.1 *Not determined

EXAMPLE 4 Optimization of CCL17 Antibodies

C17B234 and C17B240 anti-CCL17 antibodies contained a potential N-linkedglycosylation site at the beginning of the LCDR2 (“NAS”). The asparagineresidue (N) at residue position 50 (Kabat numbering) of C17B234 wasmutated to six different amino acids (A, D, G, S, T and I).

A potential aspartic acid isomerization motif, “OS” was identified inthe HCDR3 in the parent C17F24 and all its affinity matured variants. Totest the effect of substitutions at this position, the serine residue atposition 100c (Kabat numbering) was mutated to A, T or S or the D atposition 100b was mutated to E in the heavy chain of mAb C17B234. Theresulting heavy chains were paired with the light chain of mAb C17E258.

The antibodies were expressed as IgG1 and their affinity to human andcyno CCL17 was measured. Table 6 and Table 7 shows the heavy and lightchain CDR sequences of the optimized antibodies. Table 8 shows theaffinity of the antibodies for human and cyno CCL17. Mutagenesis of N50in the light chain resulted .i.n improved binding of 2- to 100-fold.

TABLE 6 HCDR1 HCDR2 HCDR3 SEQ ID  SEQ ID SEQ ID mAb ID Sequence NO:Sequence NO: Sequence NO: C17B257 SYWIG 4 IIDPSDSDTRYSPSFQC 5VCPADVWDSFDY 6 C17B258 SYWIG 4 IIDPSDSDTRYSPSFQC 5 VCPADVWDSFDY 6C17B260 SYWIG 4 IIDPSDSDTRYSPSFQC 5 VCPADVWDSFDY 6 C17B262 SYWIG 4IIDPSDSDTRYSPSFQC 5 VCPADVWDSFDY 6 C17B263 SYWIG 4 IIDPSDSDTRYSPSFQC 5VCPADVWDSFDY 6 C17B264 SYWIG 4 IIDPSDSDTRYSPSFQC 5 VCPADVWDSFDY 6C17B293 SYWIG 4 IIDPSDSDTRYSPSFQC 5 VCPADVWDAFDY 42 C17B294 SYWIG 4IIDPSDSDTRYSPSFQC 5 VCPADVWDTFDY 43 C17B295 SYWIG 4 IIDPSDSDTRYSPSFQC 5VCPADVWESFDY 44

TABLE 7 LCDR1 LCDR2 LCDR3 SEQ SEQ SEQ ID ID ID mAb ID Sequence NO:Sequence NO: Sequence NO: C17B257 KSSQSVLLSFD 8 AASTRES 39 QQFYSVPST 28NINKLA C17B258 KSSQSVLLSFD 8 DASTRES 24 QQFYSVPST 28 NINKLA C17B260KSSQSVLLSFD 8 GASTRES 22 QQFYSVPST 28 NINKLA C17B262 KSSQSVLLSFD 8SASTRES 40 QQFYSVPST 28 NINKLA C17B263 KSSQSVLLSFD 8 TASTRES 26QQFYSVPST 28 NINKLA C17B264 KSSQSVLLSFD 8 IASTRES 41 QQFYSVPST 28 NINKLAC17B293 KSSQSVLLSFD 8 DASTRES 24 QQFYSVPST 28 NINKLA C17B294 KSSQSVLLSFD8 DASTRES 24 QQFYSVPST 28 NINKLA C17B295 KSSQSVLLSFD 8 DASTRES 24QQFYSVPST 28 NINKLA

TABLE 8 Affinity (pM) Human Cyno Fold binding mAb ID CCL17 CCL17human/cyno c17B257 0.1 65.6 656.0 C17B258 0.1 41.6 416.0 C17B260 0.536.4 72.8 C17B262 0.3 55.5 185.0 C17B263 0.1 75.59 755.9 C17B264 0.471.83 179.6 C17B293 <0.1 39 C17B294 1 22 22.0 C17B295 175 29892 170.8

A Tryptophan residue in HCDR3 at position 100a (Kabat numbering) in mAbof C17B236 was identified as a putative site for undesired posttranslational oxidation. This residue was mutated to 17 other aminoacids (all except C and M) in the Fab parent of C17B236, C17F319 whichmAb is this. Kunkel's mutagenesis was performed with “NNK” or definedcodon oligonucleotides to produce this panel. These Fabs were thenscreened using a ranking ELISA for binding to both bt-human and bt-cynoCCL17. Five variants (W→R, Y, F, T, I) showed some binding in a Fabbinding ELISA (Table 5). The best three were converted to mAb (M17B288,C17B289, C17B290) for expression and MSD-SEA (Table 6). Most variantsdisplayed reduced affinity to the human and cyno CCL17. VH and VLsequences of the antibodies are shown in Table 9.

TABLE 9 VH SEQ ID VL SEQ ID mAb NO: NO: C17F24 45 49 (parent) C17B234 4550 C17B235 45 51 C17B236 45 52 C17B237 45 53 C17B238 45 54 C17B239 45 55C17B240 45 56 C17B241 45 57 C17B242 45 58 C17B243 45 59 C17B244 45 60C17B257 45 61 C17B258 45 62 C17B260 45 63 C17B262 45 64 C17B263 45 65C17B264 45 66 C17B293 46 62 C17B294 47 62 C17B295 48 62

EXAMPLE 5 Selection of Constant Regions

Select antibodies were cloned as IgG2 or IgG4 with followingsubstitutions: IgG4 S228P/L234A/L235A or IgG2V234A/G237A/P238S/H268A/V309L/A330S/P331S using standard methods. Table10 shows the resulting antibodies

TABLE 10 Variable mAb regions name isotype from mAb C17B302 IgG2 C17B293V234A/G237A/P238S/H268A/V309L/ A330S/P331S C17B311 IgG4S228P/L234A/L235A C17B293 C17B301 IgG2 C17B294V234A/G237A/P238S/H268A/V309L/ A330S/P331S C17B312 IgG4S228P/L234A/L235A C17B294Heavy and light chains of certain antibodies are shown below:

CB30 HC (SEQ ID NO: 67)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVGPADVWDAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CB302 LC (SEQ ID NO: 68)DIVMTQSPDSLAVSLGERATINCKSSQSVLLSFDNINKLAWYQQKPGQPPKLLIYDASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYSVPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECCB301 HC (SEQ ID NO: 69)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVGPADVWDTFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CB301 LC (SEQ ID NO: 70)DIVMTQSPDSLAVSLGERATINCKSSQSVLLSFDNINKLAWYQQKPGQPPKLLIYDASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYSVPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

EXAMPLE 6 Characterization of Anti-CCL17 Antibodies

Select anti-CCL17 antibodies were characterized in calcium flux,β-arrestin reporter assays and in chemotaxis assay to assess theirability to inhibit CCL17 biological activities.

Calcium flux assay. Calcium mobilization assay was used to test forability of hybridoma mAbs to neutralize CCL17 signaling. CCRF-CEM cells(ATCC® CCL-119™) were cultured in RPMI with GlutaMAX; 10% FBS; 10 mMHepes, 1 mM Sodium Pyruvate, 4500 mg/L glucose, and 1500 mg/ml Sodiumbicarbonate at 37° C. incubator with 5% CO2saturation. Cells werelabeled with dye using the Fluo-8 NW No Wash Calcium Assay Kit (#36315)from ABD Bioquest, Inc. Test antibodies and 10 ng/mL human CCL17 or 5ng/mL cyno CCL17 were pre-incubated with the test antibodies and themixture was added to the cells. Fluorescent signal was detected usingFDSS 6000 (Hamamatsu, Bridgewater, N.J.) to using 490 nm excitation and525 nm emission.

β-Arrestin Reporter Assay. β-arrestin assay was used to assess theability of the anti-CCL17 antibodies to neutralize CCL17 function. Assaywas performed using the PathHunter express β-arrestin assay (DiscoveRx). Briefly, ability of anti-CCL17 antibodies to inhibit CCL17-inducedβ-arresting recruitment was assessed in Hek293 cells co-expressing CCR4fused in frame with the small enzyme fragment ProLink™ and a fusionprotein of β-Arrestin and the N-terminal deletion mutant of β-gal(called enzyme acceptor or EA). Anti-CCL17 antibodies at variousconcentrations (0.15 nM-1 pM) were combined with 20 nM CCL17 and themixture was incubated at 37° C. for 20-30 minutes prior to adding theantibody-CCL17 complex to the cells. The mixture was then applied to thecells and incubated at 37° C. (95% O₂/5% CO₂) for 90 minutes. 55 μl ofdetection reagent was added per well and incubated for 60 min at roomtemperature. Samples were read on a standard luminescence plate reader,and IC₅₀ values were calculated.

Chemotaxis assay: Chemotaxis assay was used to demonstrate that theanti-CCL17 antibodies inhibit CCL17 function. The migration of the HSC-Fcells (HSC-F cells were obtained through the NIH Nonhuman PrimateReagent Resource) or CCRF-CEM cells (ATCC° CCL-119™) was assessed usinga 96-well chemotaxis chamber using 5 μm polycarbonate filter accordingto protocol described in Imai et al. 1997; Imai et al. 1999. Briefly,the lower chambers were filled with 320 μl RPMI/BSA 0.1% and 1 nM of thehuman or cyno CCL17 without or with different concentrations of theantibodies (0.125, 0.25, 0.5 1 and 10 μg/ml) and then were carefullyoverlaid with the polycarbonate membrane. Cells were washed with PBS andsuspended in RPMI/ESA 0.1% at 0.5×10 ⁶ cells/ml, and the cell suspensionwas added to the upper chambers. The chambers were incubated for 60 minin a 5% CO₂-humidified incubator at 37° C., and cells migrating acrossthe membrane into the lower chamber were determined using The CellTiter-Glo Luminescence Cell Viability.

Table 11 shows the IC50 values for the calcium flux assay. The data isan average of three independent experiments. Each mAb completelyneutralized the calcium flux induced by human or cyno CCL17 using either10 ng/ml (1.25 nM) human CCL17 or 5 ng/ml (0.625 nM) cyno CCL17 and asshown in Table 11, IC₅₀ values were roughly equivalent for each of theantibodies against both human and cyno protein.

TABLE 11 ICso(nM) mAb Human CCL17 Cyno CCL17 C17B302 0.593 0.238 C17B3110.553 0.239 C17B318 0.275 0.237 C17B319 0.753 0.289 C17B234 0.421 0.369C17B235 0.558 0.919 C17B236 0.385 0.349 C17B237 0.882 0.549 C17B2380.387 0.348 C17B239 0.427 0.430 C17B240 0.405 0.308 C17B241 0.456 0.339C17B242 0.483 0.340 C17B243 0.231 0.310 C17B244 0.311

Table 12 shows the IC₅₀ values for the β-arrestin assay. The data is anaverage of three independent experiments. All of the mAbs were able tocompletely inhibit human or cyno CCL17-induced β-arrestin recruitment at20 nM and dose-dependently inhibit β-arrestin recruitment induced byhuman or cyno CCL17 with equivalent potency.

TABLE 12 Human CCL17 Cyno CCL17 ICso (nM) STD ICso (nM) STD C17B30213.94 9.56 13.412 6.403 C17B311 10.65 2.89 10.324 2.569 C17B318 11.0062.886 12.141 2.294 C17B319 14.225 4.133 17.51 6.605 C17B234 8.42 5.5255.3915 0.043 C17B236 6.98 3.33 9.502 0.073

FIG. 1 and FIG. 2 show the inhibition of chemotaxis with selectantibodies in human and cyno cells, respectively. All antibodies testedinhibited both human CCL17-induced CCRF-CEM cell chemotaxis at a levelof inhibition of about 50% at an antibody concentration of 0.5 μg/ml.C17B302 and C17B311 inhibited cyno HSC-F cell chemotaxis induced by cynoCCL17 at a level of inhibition of about 50% at antibody concentration of0.5 82 g/ml.

EXAMPLE 7 Epitope Mapping of Anti-CCL17 Antibody C17B236

The binding epitope of antibody C17B236 (VH: SEQ ID NO: 45; VL: SEQ IDNO: 52) was determined by X-ray crystallography.

Human CCL17 was expressed in E. coli, isolated from inclusion bodies andrefolded. The Fab fragment of mAb C17B236 was expressed in HEK293Fcells. The CCL17: C17B236 complex was prepared by mixing at a molarratio of 1.6:1 with an excess of CCL17. The complex was then purified bysize exclusion chromatography. The complex was crystallized by thevapor-diffusion method from solution containing 20% PEG 3350 and 0.2 MK/Na tartrate. The X-ray diffraction data were collected to 1.9 Åresolution. The structure was determined by molecular replacement andrefined to an R-factor of 18.0%.

C17B236 epitope is conformational and spans 3 segments of the CCL17molecule, namely two loops (residues 21-23 and 44-45) and the C-terminalhelix (residues 60-68). The key interactions involve basic residuesArg22 and Lys23 of CCL17 and a cluster of acidic residues in HCDR2including Asp52, Asp55 and Asp57. In addition to these electrostaticinteractions, van-der-Waals contacts in the center of the epitope occurbetween Trp33 and Trp105 of VH and Arg22 of CCL17. Given the number ofcontacts, the key residue of the epitope is Arg22, which stacks againstTrp33 of VH and makes numerous contacts to HCDR3. The paratope and theepitope residues are shown in FIG. 7.

The paratope (antibody residues involved in binding CCL17) includes 18residues that belong to 5 out of 6 CDRs (all except LCDR2).

The C17B236 epitope is on the opposite side of the CCL17 monomer fromits dimerization surface. C17B236 thus does not block dimerization ofCCL17. The neutralization effect of C17B236 results from the competitionwith CCR4 for the overlapping epitopes.

Sequence Listing: SEQ ID NO Type Species Description Sequence 1 PRT HomoCCL17 argtnvgreccleyfkgai sapiens plrklktwyqtsedcsrdaivfvtvqgraicsdpnnkr vknavkylqslers 2 PRT Cyno CCL17 margtnvgrecclkyfkgaiplrklktwyqtsedcsrd aivfvtvqnkaicsdpndk kvkkalkylqslers 3 PRT cyno CCL22gpyganmedsvccrdyvry rmplrvvkhfywtsdscpr pgvvlltsrdkeicadpry pwvkmilnklsq4 PRT Artificial HCDRl SYWIG sequence fo C17F24 5 PRT Artificial HCDR2IIDPSDSDTRYSPSFQG sequence of C17F24 6 PRT Artificial HCDR3 VGPADVWDSFDYsequence of C17F24 7 PRT Artificial LCDRl KSSQSVLYSSNNKNYLA sequence ofCl7F24 (parent) 8 PRT Artificial LCDRl KSSQSVLLSFDNINKLA sequence ofCl7B234 9 PRT Artificial LCDRl KSSQSVLYSFYNFNALA sequence of Cl7B235 10PRT Artificial LCDRl KSSQSVLLSPWNSNQLA sequence of Cl7B236 11 PRTArtificial LCDRl KSSQSVLTSYNNSNYLA sequence of Cl7B237 12 PRT ArtificialLCDRl KSSQSVLISAFNQNPLA sequence of Cl7B238 13 PRT Artificial LCDRlKSSQSVLSSFTNTNTLA sequence of Cl7B239 14 PRT Artificial LCDRlKSSQSVLYSHVNYNALA sequence of Cl7B240 15 PRT Artificial LCDRlKSSQSVLNSFTNNNALA sequence of Cl7B241 16 PRT Artificial LCDRlKSSQSVLFSHDNLNTLA sequence of Cl7B242 17 PRT Artificial LCDRlKSSQSVLNSFDNKNDLA sequence of Cl7B243 18 PRT Artificial LCDR1KSSQSVLSSITNVNDLA sequence of Cl7B244 19 PRT Artificial LCDR2 WASTRESsequence of Cl7F24 (parent) 20 PRT Artificial LCDR2 NASTRES sequence ofCl7B234 21 PRT Artificial LCDR2 HASTRES sequence of Cl7B235 22 PRTArtificial LCDR2 GASTRES sequence of Cl7B236 23 PRT Artificial LCDR2LASTRES sequence fo Cl7B237 24 PRT Artificial LCDR2 DASTRES sequence ofCl7B238 25 PRT Artificial LCDR2 EASTRES sequence of Cl7B241 26 PRTArtificial LCDR2 TASTRES sequence of Cl7B244 27 PRT Artificial LCDR3QQYYSTPLT sequence of Cl7F24 (parent) 28 PRT Artificial LCDR3 QQFYSVPSTsequence of Cl7B234 29 PRT Artificial LCDR3 QQFYATPFT sequence ofCl7B235 30 PRT Artificial LCDR3 QQYYLIPST sequence of Cl7B236 31 PRTArtificial LCDR3 QQYLSPPST sequence of Cl7B237 32 PRT Artificial LCDR3QQYQFIPFT sequence of Cl7B238 33 PRT Artificial LCDR3 QQYLIYPST sequenceof Cl7B239 34 PRT Artificial LCDR3 QQYYTLPAT sequence of Cl7B240 35 PRTArtificial LCDR3 QQTNSIPLT sequence of Cl7B241 36 PRT Artificial LCDR3QQYYAVPQT sequence of Cl7B242 37 PRT Artificial LCDR3 QQHWQTPLT sequenceof Cl7B243 38 PRT Artificial LCDR3 QQYYHDPFT sequence of Cl7B244 39 PRTLCDR2 AASTRES of Cl7B257 40 PRT LCDR2 SASTRES of Cl7B262 41 PRT LCDR2IASTRES fo Cl7B264 42 PRT HCDR3 VGPADVWDAFDY of Cl7B293 43 PRT HCDR3VGPADVWDTFDY of Cl7B294 44 PRT HCDR3 VGPADVWESFDY of Cl7B295 45 PRTVH of EVQLVQSGAEVKKPGESLK C17F24, ISCKGSGYSFTSYWIGWVR C17B234,QMPGKGLEWMGIIDPSDSD C17B235, TRYSPSFQGQVTISADKSI C17B236,STAYLQWSSLKASDTAMYY C17B237, CARVGPADVWDSFDYWGQG C17B238, TLVTVSSC17B239, C17B240, C17B241, C17B242, C17B243, C17B244, C17B257, C17B258,C17B260, C17B262, C17B266, C17B264 46 PRT Homo VH ofEVQLVQSGAEVKKPGESLKISC sapiens C17M293 KGSGYSFTSYWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPSF QGQVTISADKSISTAYLQWSSL KASDTAMYYCARVGPADVWDAFDYWGQGTLVTVSS 47 PRT Homo VH of EVQLVQSGAEVKKPGESLKISC sapiensC17B294 KGSGYSFTSYWIGWVRQMPG KGLEWMGIIDPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSL KASDTAMYYCARVGPADVW DTFDYWGQGTLVTVSS 48 PRT HomoVH of EVQLVQSGAEVKKPGESLKISC sapiens C17B295 KGSGYSFTSYWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPPF QGQVTISADKSISTAYLQWSSL KASDTAMYYCARVGPADVWESFDYWGQGTLVTVSS 49 PRT Homo VL of DIVMTQSPDSLAVSLGERATINC sapiens C17F24KSSQSVLYSSNNKNYLAWYQQKP (parent) GQPPKLLIYWASTRESGVPDRFSGSGSGIDFILTISSLQAEDVAVY YCQQYYSTPLTFGQGTKVEIK 50 PRT Homo VL ofDIVMTQSPDSLAVSLGERATIN sapiens C171B234 CKSSQSVLLSFDNINKLAWYQQKPGQPPKLLIYNASTRESGVP DRFSGSGSGTDFTLTISSLQAE DVAVYYCQQFYSVPSTFGQGTKVEIK 51 PRT Homo VL of DIVMTQSPDSLAVSLGERATIN sapiens C17B235CKSSQSVLYSFYNFNALAWYQ QKPGQPPKLLIYHASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYATPFTFGQGT KVEI 52 PRT Homo VL of DIVMTQSPDSLAVSLGERATINsapiens C171B236 CKSSQSVLLSPWNSNQLAWY QQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYLIPSTFGQGTK VEIK 53 PRT Homo VL ofDIVMTQSPDSLAVSLGERATIN sapiens C17B237 CKSSQSVLTSYNNSNYLAWYQQKPGQPPKLLIYLASTRESGVP DRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYLSPPSTFGQGTKVEIK 54 PRT Homo VL of DIVMTQSPDSLAVSLGERATIN sapiens C17B238CKSSQSVLISAFNQNPLAWYQ QKPGQPPKLLIYDASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYQFIPFTFGQGT KVEIK 55 PRT Homo VL of DIVMTQSPDSLAVSLGERATINsapiens C17B229 CKSSQSVLSSFTNTNTLAWYQ QKPGQPPKLLIYHASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYLIYPSTFGQGTK VEIK 56 PRT Homo VL ofDIVMTQSPDSLAVSLGERATIN sapiens C17B240 CKSSQSVLYSHVNYNALAWYQQKPGQPPKLLIYNASTRESGVP DRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYTLPATFGQGTKVEIK 57 PRT Homo VL of DIVMTQSPDSLAVSLGERATIN sapiens C171B241CKSSQSVLNSFTNNNALAWY QQKPGQPPKLLIYEASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTNSIPLTFGQGT KVEIK 58 PRT Homo VL of DIVMTQSPDSLAVSLGERATINsapiens C17B242 CKSSQSVLFSHDNLNTLAWYQ QKPGQPPKLLIYHASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYAVPQTFGQG TKVEIK 59 PRT Homo VL ofDIVMTQSPDSLAVSLGERATIN sapiens C17B243 CKSSQSVLNSFDNKNDLAWYQQKPGQPPKLLIYEASTRESGV PDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQHWQTPLTFGQGTKVEIK 60 PRT Homo VL of DIVMTQSPDSLAVSLGERATINC sapiens C17B244KSSQSVLSSITNVNDLAWYQQKP GQPPKLLIYTASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAVYYCQQYYHDPFTFGQGTKVEIK 61 PRT Homo VL of DIVMTQPDSLAVSLGERATIN sapiensC17B257 CKSSQSVLLSFDNINKLAWYQ QKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQFYSVPSTFGQGT KVEIK 62 PRT Homo VL ofDIVMTQSPDSLAVSLGERATIN sapiens C17B258 CKSSQSVLLSFDNINKLAWYQQKPGQPPKLLIYDASTRESGVP DRFSGSGSGTDFTLTISSLQAE DVAVYYCQQFYSVPSTFGQGTKVEIK 63 PRT Homo VL of DIVMTQSPDSLAVSLGERATIN sapiens C17B260CKSSQSVLLSFDNINKLAWYQ QKPGQPPKLLIYGASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYSVPSTFGQGT KVEIK 64 PRT Homo VL of DIVMTQSPDSLAVSLGERATINsapiens C17B262 CKSSQSVLLSFDNINKLAWYQ QKPGQPPKLLIYSASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQFYSVPSTFGQGT KVEIK 65 PRT Homo VL ofDIVMTQSPDSLAVSLGERATIN sapiens C17B263 CKSSQSVILSFDNINKLAWYQQKPGQPPKLLIYTASTRESGVP DRFSGSGSGTDFILTISSLQAE DVAVYYCQFYSVPSTFGQGT KVEIK66 PRT Homo VL of DIVMTQSPDSLAVSLGERATIN sapiens C17B264CKSSQSVLLSFDNINKLAWYQ QKPGQPPKLLIYTASTRESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYSVPSTFGQGT KVEIK 67 PRT Homo CB302HC EVQLVQSGAEVKKPGESLKsapiens ISCKGSGYSFTSYWIGWVR QMPGKGLEWMGIIDPSDSD TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYY CARVGPADVWDAFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHK PSNTKVDKTVERKCCVECP PCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDV SAEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKTKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 68 PRT Homo CB302LC DIVMTQSPDSLAVSLGERAsapiens TINCKSSQSVLLSFDNINK LAWYQQKPGQPPKLLIYDA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ QFYSVPSTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 69 PRT Homo CB301HC EVQLVQSGAEVKKPGESLKsapiens ISCKGSGYSFTSYWIGWVR QMPGKGLEWMGIIDPSDSD TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYY CARVGPADVWDTFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHK PSNTKVDKTVERKCCVECP PCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDV SAEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKTKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 70 PRT Homo CB301LC DIVMTQSPDSLAVSLGERAsapiens TINCKSSQSVLLSFDNINK LAWYQQKPGQPPKLLIYDA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ QFYSVPSTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

1-31. (canceled)
 32. An isolated polynucleotide encoding an antibodythat specifically binds human CCL17, wherein the polynucleotide encodesa heavy chain complementarity determining region (HCDR) 1, a HCDR2, anda HCDR3, and a light chain complementarity determining region (LCDR) 1,a LCDR2, and a LCDR3 of SEQ ID NOs: 4, 5, 42, 8, 24 and 28,respectively.
 33. The isolated polynucleotide of claim 32, wherein thepolynucleotide encodes a heavy chain variable region (VH) of SEQ ID NO:46 and a light chain variable region (VL) of SEQ ID NO:
 62. 34. A vectorcomprising the isolated polynucleotide of claim
 32. 35. A host cellcomprising the vector of claim
 33. 36. A method of producing anantibody, comprising culturing the host cell of claim 34 in conditionssuch that the antibody is produced.
 37. The isolated polynucleotide ofclaim 32, wherein the polynucleotide comprises one or more regulatoryelements.
 38. The isolated polynucleotide of claim 37, wherein theregulatory element includes an enhancer or promoter.
 39. The isolatedpolynucleotide of claim 32, wherein the encoded antibody is human orhumanized.
 40. The isolated polynucleotide of claim 32, wherein theencoded antibody is of IgG1, IgG2, IgG3, or IgG4 isotype.
 41. Theisolated polynucleotide of claim 32, wherein the encoded antibodycomprises a substitution in an Fc region.
 42. The isolatedpolynucleotide of claim 41, wherein the substitution comprises V234A,G237A, P238S, H268A, V309L, A330S, or P331S substitution on IgG2, orS228P, L234A, or L235A substitution on IgG4, wherein residue numberingis according to the EU Index.
 43. The isolated polynucleotide of claim42, wherein the substitution comprisesV234A/G237A/P238S/H268A/V309L/A330S/P331S substitution on IgG2 orS228P/L234A/L235A substitution on IgG4, wherein residue numbering isaccording to the EU Index.